Glucose oxidase compositions as a neonate anticonvulsant

ABSTRACT

Neonatal seizure is different from adult seizure, and many anti epileptic drugs that are effective in adults often fail to treat neonatal seizure. Gluconic acid, a natural organic acid enriched in fruits and honey, and the glucose oxidase enzyme, is shown herein to potently inhibit neonatal epilepsy both in vitro and in vivo. Sodium gluconate is shown to inhibit epileptiform burst activity in cell cultures and protect neurons from kainic acid-induced cell death. Sodium gluconate also inhibited epileptiform burst activity in brain slices in a manner that was much more potent in neonatal animals than in older animals. Consistently, in vivo EEC recordings also revealed that sodium gluconate inhibited the epileptic seizure activity in a manner that was much more potent in neonates than in adult animals. Mechanistically, sodium gluconate inhibits voltage-dependent CLC-3 C1− channels both in neuronal cultures and in hippocampal slices. Together, these data suggest a novel antiepileptic drug gluconate that potently inhibits neonatal seizures through blocking CLC-3 C1− channels.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Grant Nos.MH083911 and AG045656, awarded by The National Institutes of Health. TheGovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention is related to the field of neurological disorders.In particular, the prevention and treatment of convulsive disordersincluding, but not limited to, muscular tonic/clonic convulsions,epilepsy, Jacksonian disorders and/or involuntary tremors. For example,some embodiments are directed to treating and preventing convulsivedisorders in neonates and/or infants. Gluconate-based and glucoseoxidase compositions have been found to be selectively effective intreating and/or preventing convulsive disorders in neonates and/orinfants.

BACKGROUND

The incidence of epilepsy is highest in the first year of life with areported incidence around 1.8-3.5/1,000 live births in the UnitedStates. Silverstein et al., “Neonatal seizures” Annals Of Neurology62:112-120 (2007); and Jensen F., “Neonatal seizures: An update onmechanisms and management” Clinics In Perinatology 36:881-900 (2009).Although many antiepileptic drugs (AEDs) have been developed over thepast several decades, neonatal seizures are often refractory to currentAEDs, which are more effective in older children or adults. Painter etal., “Phenobarbital compared with phenytoin for the treatment ofneonatal seizures” The New England Journal Of Medicine 341:485-489(1999); van Rooij et al., “Treatment of neonatal seizures” Seminars InFetal &Neonatal Medicine 18:209-215 (2013); and Dzhala et al., “NKCC1transporter facilitates seizures in the developing brain” NatureMedicine 11:1205-1213 (2005).

In some cases, even after AED application, electroencephalographic (EEG)recordings show ongoing cortical epileptic activity in neonates, whichmay impair cognitive development and later result in epilepsy. Connellet al., “Clinical and EEG response to anticonvulsants in neonatalseizures” Archives Of Disease In Childhood 64:459-464 (1989); Glykys etal., “Differences in cortical versus subcortical GABAergic signaling: acandidate mechanism of electroclinical uncoupling of neonatal seizures”Neuron 63:657-672 (2009); and Puskajov et al., “Pharmacotherapeutictargeting of cation-chloride cotransporters in neonatal seizures”Epilepsia 55:806-818 (2014).

Epileptic seizures are often caused by overexcitation of the braincircuits, which can be inhibited by boosting GABA_(A) receptors(GABA_(A)-Rs), the major inhibitory receptors in the adult brain.Accordingly, AEDs are often developed to increase GABA_(A)-R function,such as benzodiazepine and barbiturate drugs. Bialer, M. & White, H. S.Key factors in the discovery and development of new antiepileptic drugs.Nature reviews. Drug discovery 9, 68-82 (2010). However, whileGABA_(A)-Rs are mostly inhibitory in the adult brain, they areexcitatory in the developing brain. Chen, G., Trombley, P. Q. & van denPol, A. N. Excitatory actions of GABA in developing rat hypothalamicneurones. The Journal of physiology 494 (Pt 2), 451-464 (1996); andBen-Ari, Y. Excitatory actions of gaba during development: the nature ofthe nurture. Nature reviews. Neuroscience 3, 728-739 (2002).

The excitatory GABAergic transmission in the developing brain alsoexplains why GABA agonists are often ineffective in controlling neonatalseizures, and sometimes can even exacerbate neonatal seizure activity.Farwell, J. R., et al. Phenobarbital for febrile seizures-effects onintelligence and on seizure recurrence. The New England journal ofmedicine 322, 364-369 (1990).

Classically, GABA excitatory versus inhibitory function has beenattributed to the regulation by Cl⁻ co-transporters NKCC1 and KCC2. 10.Kaila, K., Price, T. J., Payne, J. A., Puskarjov, M. & Voipio, J.Cation-chloride cotransporters in neuronal development, plasticity anddisease. Nature reviews. Neuroscience 15, 637-654 (2014). Blaesse, P.,Airaksinen, M. S., Rivera, C. & Kaila, K. Cation-chloride cotransportersand neuronal function. Neuron 61, 820-838 (2009). Previous study foundthat NKCC1 might facilitate neonatal seizures in rodent animals¹², butrecent clinical trial in infant babies found severe side effect of NKCCblocker bumetanide and very limited effect in treating neonatalseizure”. Dzhala, V. I., et al. NKCC1 transporter facilitates seizuresin the developing brain. Nature medicine 11, 1205-1213 (2005); andPressler, R. M., et al. Bumetanide for the treatment of seizures innewborn babies with hypoxic ischaemic encephalopathy (NEMO): anopen-label, dose finding, and feasibility phase 1/2 trial. Lancet Neurol14, 469-477 (2015).

Unfortunately, to date there have been no effective drugs that can treatneonatal seizures successfully, prompting an urgent search of new drugsfor neonatal epilepsy. What is needed in the art is a simple, effectiveand safe anti-epileptic drug to treat the unique characteristics ofneonatal seizure.

SUMMARY OF THE INVENTION

The present invention is related to the field of neurological disorders.In particular, the prevention and treatment of convulsive disordersincluding, but not limited to, muscular tonic/clonic convulsions,epilepsy, Jacksonian disorders and/or involuntary tremors. For example,some embodiments are directed to treating and preventing convulsivedisorders in neonates and/or infants. Gluconate-based and glucoseoxidase compositions have been found to be selectively effective intreating and/or preventing convulsive disorders in neonates and/orinfants.

In one embodiment, the present invention contemplates a method,comprising: a) providing; i) a neonatal patient comprising glucose andexhibiting convulsions; and ii) a composition comprising glucoseoxidase; and b) administering said composition to said patient underconditions such that said convulsions are reduced. In one embodiment,said conditions comprise the conversion of said glucose into gluconate.In one embodiment, the administering further comprises supplementaryglucose. In one embodiment, the composition further comprisessupplementary glucose. In one embodiment, said composition and saidsupplementary glucose are administered in series. In one embodiment,said composition and said supplementary glucose are administeredsimultaneously. In one embodiment, said conditions comprise theconversion of said supplementary glucose into gluconate. In oneembodiment, said composition is a pharmaceutically acceptablecomposition. In one embodiment, said administering further comprises aneffective amount of said composition.

In one embodiment, the present invention contemplates a method,comprising: a) providing: i) a neonatal patient exhibiting convulsions,wherein said patient is not in need of a divalent cation-based therapy;and ii) a composition comprising a gluconate complex; and b)administering said composition to said patient under conditions suchthat said convulsions are reduced. In one embodiment, the gluconatecomplex lacks a divalent cation. In one embodiment, the gluconatecomplex is sodium gluconate. In one embodiment, the divalentcation-based therapy is a calcium-based therapy. In one embodiment, thedivalent cation-based therapy is a magnesium-based therapy. In oneembodiment, the divalent cation-based therapy is a zinc-based therapy.In one embodiment, the gluconate complex comprises a gluconic acidderivative.

In one embodiment, the present invention contemplates a method,comprising: a) providing: i) a neonatal patient exhibiting convulsions,wherein said patient is not in need of a divalent cation-based therapy;ii) a composition consisting of a gluconate complex, wherein saidcomplex lacks a divalent cation; and b) administering said compositionto said patient under conditions such that said convulsions are reduced.In one embodiment, the gluconate complex is sodium gluconate. In oneembodiment, the divalent cation-based therapy is a calcium-basedtherapy. In one embodiment, the divalent cation-based therapy is amagnesium-based therapy. In one embodiment, the divalent cation-basedtherapy is a zinc-based therapy. In one embodiment, the gluconatecomplex comprises a gluconic acid derivative.

In one embodiment, the present invention contemplates a compositioncomprising a gluconate derivative wherein at least one hydroxyl moietyor carboxylic acid moiety is substituted with a group including, but notlimited to, a substituted or unsubstituted aryl or heteroaryls, anunsubstituted or substituted C1-C6-alkyl group, a substituted orunsubstituted 5-6-membered saturated or unsaturated fused ring, asubstituted or unsubstituted 5-6-membered saturated or non-saturatedring, natural amino acid residues or synthetic amino acid residues,trihalomethyl, substituted or unsubstituted C1-C6-alkoxy, NH₂, SH,thioalkyl, aminoacyl, aminocarbonyl, substituted or unsubstitutedC1-C6-alkoxycarbonyl, aryl, heteroaryl, substituted or unsubstituted4-8-membered cyclic alkyl, optionally containing 1-3 heteroatoms,carboxyl, cyano, halogen, hydroxy, nitro, acetoxy, aminoacyl, sulfoxy,sulfonyl, C1-C6-thioalkoxy, C1-C6-aliphatic alkyl, substituted orunsubstituted saturated cyclic C4-C8-alkyl optionally containing 1-3heteroatoms and optionally fused with an aryl or an heteroaryl; asubstituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, whereby said aryl or heteroaryl groups are optionallysubstituted with substituted or unsubstituted C1-C6-alkyl, liketrihalomethyl, substituted or unsubstituted C1-C6-alkoxy, substituted orunsubstituted C2-C6-alkenyl, substituted or unsubstituted C2-C6-alkynyl,amino, aminoacyl, aminocarbonyl, substituted or unsubstitutedC1-C6-alkoxycarbonyl, aryl, carboxyl, cyano, halogen, hydroxy, nitro,acetoxy, aminoacyl, sulfoxy, sulfonyl, C1-C6-thioalkoxy; or R5 and R6taken together could form a substituted or unsubstituted 4-8-memberedsaturated cyclic alkyl or heteroalkyl group.

In one embodiment, the present invention contemplates a methodcomprising: a) providing; i) a neonatal patient exhibiting convulsions,wherein said patient comprises at least one chloride ion channel; ii) acomposition comprising a chloride ion channel blocker; and b)administering said composition to said patient under conditions suchthat said convulsions are reduced. In one embodiment, the chloride ionchannel blocker is a gluconate complex that lacks a divalent cation. Inone embodiment, the chloride ion channel blocker includes, but is notlimited to, niflumic acid (NFA), flufenamic acid,4,4′-Diisothiocyanato-2,2′-stilbenedisulfonic acid disodium salt (DIDS),and 5-Nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). In oneembodiment, the gluconate complex is sodium gluconate. In oneembodiment, the patient is not in need of a divalent-cation basedtherapy. In one embodiment, the divalent cation-based therapy is acalcium-based therapy. In one embodiment, the divalent cation-basedtherapy is a magnesium-based therapy. In one embodiment, the divalentcation-based therapy is a zinc-based therapy. In one embodiment, thegluconate complex comprises a gluconic acid derivative.

Definitions

To facilitate the understanding of this invention, a number of terms aredefined below. Terms defined herein have meanings as commonly understoodby a person of ordinary skill in the areas relevant to the presentinvention. Terms such as “a”, “an” and “the” are not intended to referto only a singular entity but also plural entities and also includes thegeneral class of which a specific example may be used for illustration.The terminology herein is used to describe specific embodiments of theinvention, but their usage does not delimit the invention, except asoutlined in the claims.

The term “gluconic acid” or “gluconate” as used herein, refers to a sixα-carbon chain comprising five hydroxyl moieties and on carboxylic acidmoiety. Each of the hydroxyl and carboxylic acid moieties may besubstituted and/or derivatized as disclosed herein to provideanti-convulsant gluconate derivatives that have improved clinicaleffectiveness as compared to traditional divalent cation therapies.

The term “convulsion” or “seizure” as used herein, refers to anyabnormal violent and involuntary contraction or series of contractionsof the muscles and/or physical manifestations including, but not limitedto, convulsions, sensory disturbances, and/or loss of consciousness,resulting from abnormal electrical discharges in the brain.

The term “neonate” or “neonatal” as used herein, refers to any new borninfant, generally less than one month old. Generally, reference toneonate age is characterized by “N”, where “P” refers to postnatal, and“#” indicates days-old, where P0 is the day of birth.

The term “divalent cation” refers to any ion carrying a valence chargeof positive two (e.g., 2+). For example, the ions Ca²⁺, Mg²⁺ and/or Zn²⁺are considered to be divalent cations.

The term “about” as used herein, in the context of any of any assaymeasurements refers to +/−5% of a given measurement.

The term “effective amount” as used herein, refers to a particularamount of a pharmaceutical composition comprising a therapeutic agentthat achieves a clinically beneficial result (i.e., for example, areduction of symptoms). Toxicity and therapeutic efficacy of suchcompositions can be determined by standard pharmaceutical procedures incell cultures or experimental animals, e.g., for determining the LD₅₀(the dose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index, and itcan be expressed as the ratio LD₅₀/ED₅₀. Compounds that exhibit largetherapeutic indices are preferred. The data obtained from these cellculture assays and additional animal studies can be used in formulatinga range of dosage for human use. The dosage of such compounds liespreferably within a range of circulating concentrations that include theED₅₀ with little or no toxicity. The dosage varies within this rangedepending upon the dosage form employed, sensitivity of the patient, andthe route of administration.

The term “symptom”, as used herein, refers to any subjective orobjective evidence of disease or physical disturbance observed by thepatient. For example, subjective evidence is usually based upon patientself-reporting and may include, but is not limited to, pain, headache,visual disturbances, nausea and/or vomiting. Alternatively, objectiveevidence is usually a result of medical testing including, but notlimited to, body temperature, complete blood count, lipid panels,thyroid panels, blood pressure, heart rate, electrocardiogram, tissueand/or body imaging scans.

The term “disease” or “medical condition”, as used herein, refers to anyimpairment of the normal state of the living animal or plant body or oneof its parts that interrupts or modifies the performance of the vitalfunctions. Typically manifested by distinguishing signs and symptoms, itis usually a response to: i) environmental factors (as malnutrition,industrial hazards, or climate); ii) specific infective agents (asworms, bacteria, or viruses); iii) inherent defects of the organism (asgenetic anomalies); and/or iv) combinations of these factors.

The terms “reduce,” “inhibit,” “diminish,” “suppress,” “decrease,”“prevent” and grammatical equivalents (including “lower,” “smaller,”etc.) when in reference to the expression of any symptom in an untreatedsubject relative to a treated subject, mean that the quantity and/ormagnitude of the symptoms in the treated subject is lower than in theuntreated subject by any amount that is recognized as clinicallyrelevant by any medically trained personnel. In one embodiment, thequantity and/or magnitude of the symptoms in the treated subject is atleast 10% lower than, at least 25% lower than, at least 50% lower than,at least 75% lower than, and/or at least 90% lower than the quantityand/or magnitude of the symptoms in the untreated subject.

The term “attached” as used herein, refers to any interaction between amedium (or carrier) and a drug. Attachment may be reversible orirreversible. Such attachment includes, but is not limited to, covalentbonding, ionic bonding, Van der Waals forces or friction, and the like.A drug is attached to a medium (or carrier) if it is impregnated,incorporated, coated, in suspension with, in solution with, mixed with,etc.

The term “medium” as used herein, refers to any material, or combinationof materials, which serve as a carrier or vehicle for delivering of adrug to a treatment point (e.g., wound, surgical site etc.). For allpractical purposes, therefore, the term “medium” is consideredsynonymous with the term “carrier”. It should be recognized by thosehaving skill in the art that a medium comprises a carrier, wherein saidcarrier is attached to a drug or drug and said medium facilitatesdelivery of said carrier to a treatment point. Further, a carrier maycomprise an attached drug wherein said carrier facilitates delivery ofsaid drug to a treatment point. Preferably, a medium is selected fromthe group including, but not limited to, foams, gels (including, but notlimited to, hydrogels), xerogels, microparticles (i.e., microspheres,liposomes, microcapsules etc.), bioadhesives, or liquids. Specificallycontemplated by the present invention is a medium comprisingcombinations of microparticles with hydrogels, bioadhesives, foams orliquids. Preferably, hydrogels, bioadhesives and foams comprise any one,or a combination of, polymers contemplated herein. Any mediumcontemplated by this invention may comprise a controlled releaseformulation. For example, in some cases a medium constitutes a drugdelivery system that provides a controlled and sustained release ofdrugs over a period of time lasting approximately from 1 day to 6months.

The term “drug” or “compound” as used herein, refers to anypharmacologically active substance capable of being administered whichachieves a desired effect. Drugs or compounds can be synthetic ornaturally occurring, non-peptide, proteins or peptides, oligonucleotidesor nucleotides, polysaccharides or sugars.

The term “administered” or “administering”, as used herein, refers toany method of providing a composition to a patient such that thecomposition has its intended effect on the patient. An exemplary methodof administering is by a direct mechanism such as, local tissueadministration (i.e., for example, extravascular placement), oralingestion, intravenous injection, transdermal patch, topical,inhalation, suppository etc.

The term “patient” or “subject”, as used herein, is a human or animaland need not be hospitalized. For example, out-patients, persons innursing homes are “patients.” A patient may comprise any age of a humanor non-human animal and therefore includes both adult and juveniles(i.e., neonates, infants and/or children). It is not intended that theterm “patient” connote a need for medical treatment, therefore, apatient may voluntarily or involuntarily be part of experimentationwhether clinical or in support of basic science studies.

The term “effective amount” as used herein, refers to a particularamount of a pharmaceutical composition comprising a therapeutic agentthat achieves a clinically beneficial result (i.e., for example, areduction of symptoms). Toxicity and therapeutic efficacy of suchcompositions can be determined by standard pharmaceutical procedures incell cultures or experimental animals, e.g., for determining the LD50(the dose lethal to 50% of the population) and the ED50 (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index, and itcan be expressed as the ratio LD50/ED50. Compounds that exhibit largetherapeutic indices are preferred. The data obtained from these cellculture assays and additional animal studies can be used in formulatinga range of dosage for human use. The dosage of such compounds liespreferably within a range of circulating concentrations that include theED50 with little or no toxicity. The dosage varies within this rangedepending upon the dosage form employed, sensitivity of the patient, andthe route of administration.

The term “pharmaceutically” or “pharmacologically acceptable”, as usedherein, refer to molecular entities and compositions that do not produceadverse, allergic, or other untoward reactions when administered to ananimal or a human.

The term, “pharmaceutically acceptable carrier”, as used herein,includes any and all solvents, or a dispersion medium including, but notlimited to, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils, coatings, isotonic and absorption delayingagents, liposome, commercially available cleansers, and the like.Supplementary bioactive ingredients also can be incorporated into suchcarriers.

The term “small organic molecule” as used herein, refers to any moleculeof a size comparable to those organic molecules generally used inpharmaceuticals. The term excludes biological macromolecules (e.g.,proteins, nucleic acids, etc.). Preferred small organic molecules rangein size from approximately 10 Da up to about 5000 Da, more preferably upto 2000 Da, and most preferably up to about 1000 Da.

The term “biologically active” refers to any molecule having structural,regulatory or biochemical functions. For example, biological activitymay be determined, for example, by restoration of wild-type growth incells lacking protein activity. Cells lacking protein activity may beproduced by many methods (i.e., for example, point mutation andframe-shift mutation). Complementation is achieved by transfecting cellswhich lack protein activity with an expression vector which expressesthe protein, a derivative thereof, or a portion thereof.

The term “transfection” or “transfected” refers to the introduction offoreign DNA into a cell.

The term “bind” as used herein, includes any physical attachment orclose association, which may be permanent or temporary. Generally, aninteraction of hydrogen bonding, hydrophobic forces, van der Waalsforces, covalent and ionic bonding etc., facilitates physical attachmentbetween the molecule of interest and the analyte being measuring. The“binding” interaction may be brief as in the situation where bindingcauses a chemical reaction to occur. That is typical when the bindingcomponent is an enzyme and the analyte is a substrate for the enzyme.Reactions resulting from contact between the binding agent and theanalyte are also within the definition of binding for the purposes ofthe present invention.

The term, “supplementary glucose” as used herein, refers to theadministration of intravenous glucose to a pediatric patient incombination with glucose oxidase. For example, a glucose infusion rate(GIR) of 7.3 mg/kg/minute is preferred.

BRIEF DESCRIPTION OF THE FIGURES

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawings will be provided by the Patentand Trademark Office upon request and payment of the necessary fee.

FIG. 1 presents exemplary data showing anti-burst activity of gluconateon cultured neurons. Data are presented as mean±s.e.m., * P<0.05, **P<0.01, *** P<0.001.

FIG. 1A: Spontaneous burst action potentials in cultured neurons wasstopped immediately by application of 20 mM NaGcA (n=10, from 3batches), the extended graphs of before, during and after NaGcAapplication was shown in the bottom.

FIG. 1B: CTZ induced robust burst activity in cultured neurons (up) andit was completely blocked by 10 mM NaGcA (bottom).

FIG. 1C: Dose-dependent inhibition of NaGcA on CTZ induced burstactivity. Black bar indicates burst frequency and gray bar indicatesburst duration (n=11 form 4 batches, paired student t-test).

FIG. 1D: Representative trace of kainic acid (1 μM for 2 hrs) inducedrobust burst activity (up) and it was also dramatically suppressed by 10mM NaGcA (bottom).

FIG. 1E: Dose-dependent response of KA induced burst activity to NaGcA(n=12 from 3 bathes, paired student t-test).

FIG. 1F: Typical trace of 4-AP (50 μM for 2 hrs) induced robust activity(up) also was obviously suppressed by 10 mM NaGcA (bottom).

FIG. 1G: Dose response of 4-AP induced burst activity to NaGcA (n=9 from3 bathes, paired student t-test).

FIG. 1H: Representative traces shown the typical recurrent epileptiformburst induced by 10 μM CTZ (Top), and lack of burst activity inco-application of CTZ and NaGcA (10 mM).

FIG. 1I(a-c): Quantified data of panel h showed that both 5 and 10 mMNaGcA chronic co-treatment, it significantly reduced the ratio ofneurons showing burst activity (FIG. 1I(a), χ2 test), burst frequency(FIG. 1I(b), oneway ANOVA) and burst duration (FIG. 11(c) and FIG.11(b), one-way ANOVA). n=22 for CTZ, n=16 for CTZ+5 mM NaGcA and n=17for CTZ+10 mM NaGcA, all data were got from 5 batches, paired studentt-test.

FIG. 1J: Live/dead assay images showed calcein-AM (green for live) andethidium homodimer-1 (red for dead) in different groups. Scale bar, 100μm.

FIG. 1K: Quantification of cell live/dead assay. Results revealed thatNaGcA has neuron protection function of against kainic acid inducedneuronal excitotoxicity, and itself without any side effect on neuronsurvival.

FIG. 2 presents exemplary data that gluconate inhibits chloride currentsin cultured cortical neurons. Data are shown as mean±s.e.m.

(a-f) NaGcA (10 mM) had no effect on Na+ currents (a,b), K+ currents(c,d), or Ca2+ currents (e, f).

(g) Typical Cl− currents recorded before (black) and after 10 mM NaGcAapplication (green).

(h) I-V curves showing a significant NaGcA inhibition on the Cl−currents (n=7 from 3 batches of cultures, paired Student's t-test).

(i,j) NPPB (100 μM), a classical Cl− channel blocker, inhibited the Cl−currents in cultured cortical neurons.

(k,l) The inhibitory effect of another common Cl− channel blocker DIDS(100 μM) on the Cl− currents in cultured cortical neurons. Note thepersistent inhibition of DIDS on the Cl− currents after washing offDIDS. (m,n) Both NPPB (m) and DIDS (n) potently inhibited theepileptiform activity induced by CTZ, supporting that Cl− channels areinvolved in epileptogenesis.

FIG. 3 illustrates exemplary data showing an age-dependent effect ofgluconate on epileptic activity in hippocampal slices. Data are shown asmean±s.e.m.

(a) Extracellular field potential recordings in the CA3 pyramidal celllayer in hippocampal slice of the neonatal (P7) mouse. Epileptic eventswas induced by Mg2+ free aCSF and inhibited in presence of 20 mM NaGcAin Mg2+ free aCSF. Expanded traces of before, during and afterapplication of NaGcA were shown on the bottom.

(b) Power spectra of epileptic activity in 5-minute time windows before,during and after NaGcA application. The amplitude of power (integrativearea under the spectrum trace) was dramatically suppressed after NaGcAapplication.

(c,d) The comparable efficacy of anti epileptiform activity of NaGcA wasexamined in P12 hippocampal slice.

(e) NaGcA has moderate anti-epileptic discharges effect in P26hippocampal slice comparing to neonatal slices. Bottom showed theextended traces of before, during and after NaGcA perfuse. (f) Poweramplitude was slightly decreased during NaGcA application and almostrecovered after wash out NaGcA.

(g) Normalized power of epileptic activity was induced by 0 Mg2+ aCSF inconsecutive 5 min windows of before, during and after NaGcA application.NaGcA dramatically reduced the power of epileptic activity in P6-8 andP10-12 groups but less effect on P21-33 group, and the control groupdata showed that there was no significantly change after extra 20 mMNaCl was added into 0 Mg2+ aCSF during 80 min extracellular fieldpotential recording.

FIG. 4 presents exemplary data showing anti-epileptic activity of NaGcAwas tested in different neonatal slice seizure models. Data are shown asmean±s.e.m.

(a) Extracellular field potential recordings in the CA3 pyramidal celllayer, epileptic activity was induced by 50 μM 4-AP. NaGcA attenuatedseizure-like discharges in response to 50 μM 4-AP. The respective tracewas enlarged in the bottom.

(b) Power spectra of epileptic activity in 5 minutes windows of before,during and after NaGcA application. The amplitude of power spectra wassignificantly suppressed after NaGcA application.

(c) Averaged power of epileptic activity, which induced by 4-AP, wasobviously reduced by perfuse of NaGcA.

(d) NaGcA remarkably suppressed the burst activity in neonatal high K+model. Bottom showed the expanded views of before, during and afterNaGcA perfuse.

(e) Power spectra amplitude was significantly reduced during NaGcAapplication.

(f) Normalized power of epileptic activity was induced by high K+ aCSFin consecutive 5 min windows before, during and after NaGcA application.NaGcA dramatically decreased the power of high K+ induced epilepticactivity.

FIG. 5 presents exemplary data showing CLC-3 channel mediated Cl⁻current in CA3 pyramidal neuron was inhibited by gluconate. Values aremean±s.e.m.

(a) Representative Cl− currents in CA3 pyramidal neuron. The Cl−currents was remarkably inhibited by 20 mM NaGcA application (green).

(b) I-V curves plotted significant outward rectification with thereversal potential close to 0 mV. Cl− current was dramatically reducedin present of NaGcA (green).

(c) Example Cl− current traces got from CA3 pyramidal neurons which usenormal CsCl pipette solution (left red), intracellular dialysis (10 min)with anti-ClC-3 antibody (middle orange) or control rabbit IgG (rightblack).

(d) I-V plot showed remarkable reduction after anti-ClC-3 antibodydialysis (orange) but no significant change after control IgG dialysis(black).

(e) Immunostaining images of HEK293T cell transfected with either theeGFP (Left row) or ClC-3-eGFP fusion protein plasmid (Right Row). ClC-3immunoreactivity (red) was detected strongly in ClC-3-eGFP transfectedcells (arrows) but not in non-transfected or eGFP transfected cells.Scale bar, 40 sm.

(f) Typical Cl− current in HEK cell transfected with either eGFP (top)or ClC-3-eGFP plasmid (bottom), and the response to the 20 mM NaGcA(middle green).

(g) I-V relationships of before, during and wash out NaGcA forClC-3-eGFP (right, n=7 from two batches) infected cells, ClC-3 mediatedcurrent was significantly inhibited by NaGcA.

FIG. 6 presents exemplary data showing that CLC-3 was up-regulated inneonatal epileptic slices. Data are presented as mean±s.e.m., ***P<0.001.

(a) Comparable expression level of ClC-3 on hippocampal CA3, ClC-3immunoreactivity was increased after slice incubation in 0 Mg2+ aCSF for1 hr. Asterisk indicated an enlarged typical neuron with CLC-3immuno-signal (red) overlapped with phase in CA3 area. Scale bar=20 μm(5 μm in enlarged images).

(b) Quantified data of ClC-3 immunoreactivity intensity showedsignificant increase after the slice incubation in 0 Mg2+ aCSF for 1 hr(n=10 slices from 4 pups for control, n=11 slices from 4 pups for 0Mg2+, student's t-test).

(c) Western blot of hippocampal ClC-3 protein from control and 0 Mg2+aCSF (1 hr) treated slice.

(d) Quantified data showed significant increase of CLC-3 protein afterhippocampal slices treated with 0 Mg2+ aCSF (n=6 pups for each of group,student's t-test).

(e) Representative Cl− currents traces of control, Mg2+ incubation for 1hr and Mg2+ incubation for 1 hr then recorded in 20 mM NaGcA.

(f) Averaged I-V plot of Cl− currents showed significant increase in 0Mg2+ group and it was also remarkably inhibited by NaGcA.

FIG. 7 A-G presents exemplary data showing that gluconate is aneffective therapy for KA induced neonatal seizure.

FIG. 7 (a) Representative EEG trace from P12 rat, which injected withsaline (0.1 ml/10 g, i.p.) 10 min after KA injection (2 mg/kg, i.p.),showed recurrent epileptic burst discharges.

FIG. 7 (b) Expanded view of a single cluster-like epileptic dischargesfrom box b in FIG. 7a showing typical interictal, ictal-tonic andictal-clonic traces.

FIG. 7 (c) Example EEG of P12 rat, which injected with NaGcA (2 g/kg,ip) 10 min after KA injection (2 mg/kg, i.p.), showing increase in spikeactivity but no robust burst discharges was developed after NaGcAadministration. Scale bar same as in FIG. 7 a.

FIG. 7 (d) Enlarged area of box d in FIG. 7c . Scale bar same as in FIG.7b . A single spike discharge was observed in the expanded trace ascompared to two flanking baseline traces. Scale bar same as in FIG. 7 c.

FIG. 7 (e) Individual EEG power in 1 min time window of panel a (black)and d (green).

FIG. 7 (f) Group averaged EEG power in 1 min time window of saline(black, P10-12, n=11) and NaGcA administration (green, P10-12, n=11).The black arrowhead indicates the KA injection point, the red arrowheadindicates either saline or NaGcA injection point. The average power ofepileptic activity induced by KA injection was 70% lower in the presenceof NaGcA.

FIG. 7 (g) Work model of our study. Cl− influx through chloride channelswill facilitate synchronized network activity (left). But this processis suppressed by gluconic acid (GA) due to its inhibition on chloridechannels (right).

FIG. 8 presents exemplary data showing the effect of gluconate on adultepilepsy.

(a-c) Representative EEG recordings showing epileptiform activityinduced by injection of pentylenetetrazol (PTZ) (50 mg/kg, i.p.)followed by saline (a), NaGcA at 1 g/kg (b), or NaGcA at 2 g/kg (c).Open triangles indicate the first spike, and the black small arrowsindicate the first burst.

(d,e) Bar graphs showing the dose-dependent effect of NaGcA inprolonging the latency of epileptiform spikes (d) or epileptiform bursts(e) induced by PTZ (50 mg/kg, i.p.).

(f) Bar graphs showing a mild inhibitory effect of NaGcA on thebehavioral seizure score induced by PTZ (50 mg/kg, i.p.).

(g) Bar graphs showing that NaGcA dose-dependently prolonged the latencyof seizure behavior (Racine score 3 and above).

FIG. 9 presents exemplary data showing the effects of a variety ofchloride ion channel inhibitors:

(a) Niflumic acid (NFA) (100 μM)

(b) Flufenamic acid (100 μM)

(c) DIDS (100 μM)

(d) NPPB (100 μM)

FIG. 10 presents exemplary data showing the effects of a variety ofchloride ion channel inhibitors on epileptic activity in neonatalhippocampal slices:

(a) Sodium Gluconic Acid (20 mM) (NaGcA)

(b) NFA (100 μm)

(c) NPPB (100 μm)

FIG. 11 presents exemplary data showing a developmental change of CLC-3channel-mediated Cl− currents.

FIG. 11A: Representative Cl− currents recorded in CA3 pyramidal neuronsfrom mouse hippocampal slices obtained at different ages of animals (toprow, black traces). Green traces in the middle row show Cl− currentsafter inhibition by 20 mM NaGcA. The bottom row shows the I-V curves ofCl− currents in control and after NaGcA treatment in different ages ofmice.

FIG. 11B: Quantified Cl− current density in control (black) or afterNaGcA treatment (green) (HP=+90 mV). Note a significant decrease of Cl−current density in adult animals. *** P<0.001.

FIG. 11C: Dose response curve of NaGcA inhibition on Cl− currents.

FIG. 11D: Typical Cl− current traces recorded under control (black),anti-CLC-3 antibody (orange), or pre-absorbed control antibody (grey).I-V curves showing a remarkable reduction of the Cl− currents afteranti-CLC-3 antibody dialysis (orange).

FIG. 11E: Immunostaining confirming the lack of CLC-3 signal in CLC-3knockout mice. Scale bar, 10 μm.

FIG. 11F: The voltage-dependent outward rectifying Cl− currents waslargely absent in CLC-3 KO mice, supporting that CLC-3 channels mediatethe Cl⁻ currents.

FIG. 11G: I-V curve showing little inhibition of NaGcA on the remainingsmall Cl− currents in CLC-3 KO mice, supporting that NaGcA is aninhibitor of CLC-3 channels.

FIG. 11H: Expression of CLC-3 Cl− channels in HEK293T cells. Scale bar,40 μm.

FIG. 11I: Large Cl− currents recorded from HEK293T cells expressingCLC-3 channels (bottom row). Application of 20 mM NaGcA significantlyinhibited the CLC-3 channel-mediated Cl− currents (green traces).

FIG. 11J: I-V curves showing the NaGcA inhibition of CLC-3channel-mediated Cl− currents (CLC-3, 925±124 pA, n=7; CLC-3+NaGcA,544±59 pA, n=7; P<0.004, paired t-test; HP=+90 mV). Values aremean±s.e.m. FIG. 12 presents exemplary data showing the relationshipbetween CLC-3 chloride channels and neonatal epileptogenesis.

FIG. 12A: Upregulation of CLC-3 channel expression (red) in hippocampalCA3 neurons after induction of epileptiform activity in 0 Mg2+artificial cerebrospinal fluid (aCSF) (1 hr). Scale bar=5 μm.

FIG. 12B: Quantified CLC-3 immuno-intensity in control and 0 Mg²⁺ aCSF(control, n=10 slices from 4 pups; 0 Mg²⁺, n=11 slices from 4 pups;p<0.05, Student's t-test).

FIGS. 12C and 12D: Western blot analysis also showed a significantincrease of CLC-3 protein level in the hippocampus after treatment with0 Mg²⁺ aCSF (n=6 pups for both groups, p<0.001, Student's t-test).

FIG. 12E: Representative voltage-dependent Cl− current traces incontrol, 0 Mg²⁺ (1 hr), and 0 Mg²⁺+20 mM NaGcA (1 hr) conditions.

FIG. 12F: I-V curves showing a significant increase of outwardrectifying Cl⁻ currents in 0 Mg²⁺. aCSF group (red) and a remarkableinhibition by NaGcA (green).

FIG. 12G: Extracellular field potential recording showing epileptiformactivity induced by 0 Mg²⁺ aCSF and its strong inhibition by 20 mM NaGcAin the CA3 pyramidal layer in hippocampal slices of a neonatal mouse(postnatal day 7, P7).

FIG. 12H: Power spectra of epileptiform activity (5-minute time windows)before (red), during (green), and after (black) NaGcA application. Theamplitude of power (integrative area under the power spectrum trace) wassignificantly reduced after NaGcA application.

FIGS. 12I and 12J: NaGcA also showed strong efficacy ofanti-epileptiform activity in P12 hippocampal slices.

FIGS. 12K and 12L: In P26 hippocampal slices, however, NaGcA only showedmodest effect on the epileptiform activity.

FIG. 12M: Normalized power showing the time course of NaGcA inhibitionon the epileptiform activity induced by 0 Mg²⁺ aCSF. NaGcA dramaticallyreduced the power of epileptiform activity in the P6-8 and P10-12groups, but had much less inhibition in the P21-33 group. As a control,the grey line represents 20 mM NaCl effect on neonatal (P8-12)epileptiform activity.

FIGS. 12N and 12O: Representative traces of epileptiform activityinduced by 0 Mg²⁺ aCSF in hippocampal slices from WT, WT+20 mM NaGcA,and CLC-3 KO mice (all at P8-12). Note that epileptiform activity didnot last for long time in CLC-3 KO mice.

FIG. 12P: Summarized data showing the burst latency (left panel) andburst frequency (right panel) induced by 0 Mg²⁺ aCSF in hippocampalslices from WT, WT+20 mM NaGcA, and CLC-3 KO mice. Data are mean±s.e.m.,*P<0.05, ** P<0.01, *** P<0.001.

FIG. 13 presents exemplary data showing a broad anti-epileptic effect ofNaGcA in various epilepsy models in neonatal hippocampal slices.

FIG. 13A: Extracellular field potential recording showing epilepticactivity induced by 50 μM 4-AP in the CA3 pyramidal cell layer ofneonatal hippocampal slices, and its inhibition by NaGcA (20 mM).

FIG. 13B: Power spectra of epileptic activity in 5-minute time windowsbefore (red), during (green), and after (black) NaGcA application. Notethat NaGcA (green) significantly reduced the power amplitude.

FIG. 13C: Normalized power showing the time course of the inhibition ofNaGcA on the epileptic activity induced by 4-AP.

FIG. 13D: NaGcA (20 mM) remarkably suppressed the epileptiform activityin the high K+ epilepsy model in neonatal hippocampal slices.

FIG. 13E: Power spectra showing a significant reduction of epilepticactivity during NaGcA application.

FIG. 13F: Normalized power illustrating the time course of NaGcAinhibition on the epileptic activity induced by high K+ aCSF.

The grey lines in FIGS. 13C and 13F represent the 20 mM NaCl controleffect on neonatal (P8-12) epileptiform activity. Data are shown asmean±s.e.m.

FIG. 14 presents exemplary data showing that the CLC-3 channel blockergluconate potently inhibits neonatal seizure activity in vivo.

FIG. 14A: Representative EEG trace showing recurrent epileptic burstdischarges from a P12 rat after KA injection (2 mg/kg, i.p.), which wasfollowed by saline injection (0.1 ml/10 g, i.p.) with 10 min interval.

FIG. 14B: Expanded view of epileptic burst discharges from the box inFIG. 14A.

FIGS. 14C & 14D: Representative EEG trace (P12 rat) showing that NaGcAinjection (2 g/kg, i.p.) at 10 min after KA injection (2 mg/kg, i.p.)significantly inhibited epileptic burst discharges.

FIGS. 14E & 14F: Representative EEG trace showing a modest effect ofphenobarbital (25 mg/kg, i.p.) on epileptic burst activities in neonatalrat (P12).

FIGS. 14G & 14H: Representative EEG trace showing the effect ofbumetanide (2 mg/kg, i.p.) on epileptic burst activities in neonatal rat(P12).

FIGS. 14, 14J, 14K & 14L: In adult mice, KA injection (10 mg/kg, i.p.)also induced robust epileptic burst activities as shown in EEGrecordings (14I and 14J), but NaGcA (2 g/kg, i.p.) only showed modesteffect on KA-induced epileptic burst activities (14K and 14L).

FIG. 14M: Summarized data in neonatal animals showing the averaged EEGpower (5-min time window) before and after KA injection, followed byinjection of saline (black), NaGcA (green), phenobarbital (magenta), orbumetanide (blue). Note that NaGcA potently inhibited the power of EEGincrease induced by KA.

FIG. 14N: In adult animals, NaGcA (green) modestly inhibited the powerof EEG induced by KA.

In both FIGS. 14M and 14N, the black arrowhead indicates the KAinjection, and the red arrowhead indicates drug injection. Data aremean±s.e.m.

FIG. 15 presents exemplary data showing an acute effect of NaGcA onKA-induced epileptic activity in neonatal rats.

FIG. 15A: Typical epileptic EEG activity induced by KA through i.p.injection. After stable epileptic activity was recorded (˜1 hr), NaGcA(2 g/kg, i.p.) was administrated to the rat (green). Note that theepileptic activity was gradually reduced after NaGcA injection.

FIG. 15B: Expanded trace from a segment in FIG. 15A, showing robustepileptic burst activity.

FIG. 15C: Expanded view of EEG recording after NaGcA injection in FIG.15A.

FIG. 15D: Further expanded view of a segment in FIG. 15B, showingrecurrent burst activity.

FIG. 15E: Further expanded view of EEG after NaGcA injection in FIG.15C, showing significant inhibition of epileptic activity by NaGcA.

FIG. 15F: Summarized data showing the power of epileptic burst activitysignificantly reduced by NaGcA application (2 g/kg i.p., n=6, P10-12rat, green).

Data are mean±s.e.m.

FIG. 16 presents exemplary data showing that the activation of CLC-3channels alters intracellular Cl− homeostasis in developing neurons.

FIG. 16A: Epileptiform burst activity induced by 0 Mg2+ aCSF in theneonatal CA3 pyramidal neurons showed long-lasting membranedepolarization.

FIG. 16B: A long-lasting membrane depolarization pulse (40 mV, 10 s)mimicking the epileptiform burst activity significantly enhanced GABAA-Rcurrent induced by GABAA-R agonist isoguvacine (100 μM) undergramicidin-perforated whole cell recording. Such depolarization-inducedenhancement was absent in CLC-3 KO mice and strongly inhibited by CLC-3channel blocker NaGcA (20 mM).

FIG. 16C: Summarized data of the effect of membrane depolarization onGABAA-R current in WT, CLC-3 KO, and WT+NaGcA groups.

FIG. 16D: Gramicidin-perforated recordings revealed a positive shift inthe GABAA-R reversal potential (EGABA) in CA3 neurons after induction ofepileptiform activity in 0 Mg²⁺ aCSF.

FIG. 16E: Bath application of NKCC1 inhibitor bumetanide induced anegative shift in EGABA in normal aCSF, but did not abolish the positiveshift of EGABA induced by 0 Mg²⁺ aCSF.

FIG. 16F: KCC2 inhibitor VU0240551 had no effect on the EGABA undernormal aCSF, and showed no effect on the positive shift induced by 0Mg²⁺ aCSF.

FIG. 16G: NaGcA completely abolishes the positive EGABA shift induced by0 Mg²⁺ aCSF. Gluconate itself did not affect the EGABA at all in normalaCSF.

FIG. 16H: In CLC-3 KO mice, EGABA also did not change when treated with0 Mg2+ aCSF.

FIG. 161: Quantified data showing the EGABA changes under variousconditions in neonatal CA3 pyramidal neurons (e.g., P8-9).

FIG. 16J: Bar graphs showing EGABA changes in adult CA3 pyramidalneurons. Note that NaGcA (20 mM) did not change the EGABA shift in adultanimals.

FIG. 16K: Typical traces of cell-attached recording showing the spikeactivity induced by isoguvacine (10 μM, 30 s) in different groups inneonatal animals (e.g., P8-9).

FIG. 16L: Summarized data showing the percentage of neurons excited byisoguvacine. Note that NaGcA essentially abolished the GABA excitatoryactivity in 0 Mg²⁺ aCSF. Data are shown as mean±s.e.m., *P<0.05, **P<0.01, *** P<0.001.

FIG. 17 presents exemplary data showing that gluconic acid complexedwith various counter ions inhibit Cl− currents in P8-12 neonatal mice.Data are presented as mean±s.e.m.

FIG. 17A: Representative traces of out-rectifying Cl− currents inneonatal CA3 pyramidal neuron (black, n=12).

FIG. 17B: Typical traces of Cl− currents in presence of 20 mM NaGcA(green, n=7).

FIG. 17C: Typical traces of Cl− currents in presence of 10 mM Mg(GcA)₂.

FIG. 17D: Typical traces of Cl− currents in presence of 20 mM Gluconicacid (blue, n=6).

FIG. 17E: I-V plot showed remarkable reduction of Cl− currents densityafter bath application of gluconic acid and its salts.

FIG. 18 presents exemplary data showing that epileptiform activity inneonatal hippocampal slices is strongly inhibited by glucose oxidase.

FIG. 18A: Dose-dependent acute effect of glucose oxidase (GOx) onepileptiform activity induced by 0 Mg²⁺. aCSF (artificialcerebral-spinal fluid, with 20 mM glucose). Note that the epilepticactivity was effectively inhibited by acute application of >1 U/ml GOx.

FIG. 18B: Epileptiform activity induced by high K+ was also suppressedby 1 U/ml GOx.

FIG. 19 presents exemplary data showing an enhanced inhibition ofglucose oxidase on epileptiform activity after prolonged incubation inaCSF.

FIG. 19A: Epileptiform activity was induced by 0 Mg²⁺ aCSF with 4 mMglucose. After adding 1 U/ml GOx into 0 Mg²⁺ aCSF (4 mM glucose) andpre-incubating for 90 min at room temperature, the epileptic activitywas remarkably inhibited.

FIG. 19B. Even with 0.1 U/ml GOx that was pre-incubated in 0 Mg²⁺ aCSF(20 mM glucose) for 90 min, the epileptiform activity was alsoinhibited.

FIG. 20 presents exemplary data that glucose oxidase inhibitsepileptiform activity.

FIG. 20A: Experimental setup for testing the acute effect of GOx onepileptiform activity in the hippocampal slice. Field potentialrecording was placed in the CA3 pyramidal cell layer, the epileptiformactivity was induced by 0 Mg²⁺ aCSF. After stable epileptiform activityinduced, the GOx was added into the 0 Mg²⁺ aCSF.

FIG. 20B: Acute effect of 0.1 U/ml GOx on the 0 Mg²⁺ aCSF inducedepileptiform activity.

FIG. 20C: Acute effect of 0.3 U/ml GOx on the 0 Mg²⁺ aCSF inducedepileptiform activity.

FIG. 20D: Acute effect of 1 U/ml GOx on the 0 Mg²⁺ aCSF inducedepileptiform activity.

FIG. 20E: Acute effect of 1 U/ml GOx on 0 glucose; 0 Mg²⁺ aCSF inducedepileptiform activity. The glucose was replaced by 5 mM lactate and 3 mMpyruvate.

FIG. 20F: 0.1 U/ml GOx was added into 0 Mg²⁺ aCSF and incubated for over1 hour at room temperature then evaluated for induced epileptiformactivity.

FIG. 20G: The relative change of field potential power under differentconditions. Note that when the glucose was removed from the bath, theinhibitory effect of 1 U/ml GOx on the epileptiform activity was almosteliminated. Data are presented as mean±s.e.m.

FIG. 21 presents exemplary data showing that glucose oxidase inhibitsepileptiform activity when induced by either; i) high K+ aCSF; or ii)4-aminopyridine (4-AP) plus Mg²⁺-free aCSF.

FIG. 21A: Epileptiform activity induced by high K⁺ (8.5 mM) aCSF wassuppressed by 1 U/ml GOx.

FIG. 21B: 1 U/ml GOx inhibited 4-AP (50 μM)+0 Mg²⁺ aCSF inducedepileptiform activity.

FIG. 21C: Normalized epileptiform activity power induced by high K⁺ aCSF(purple) or 4-AP (50 μM)+0 Mg²⁺ aCSF (blue) before, during and after 1U/ml GOx application.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is related to the field of neurological disorders.In particular, the prevention and treatment of convulsive disordersincluding, but not limited to, muscular tonic/clonic convulsions,epilepsy, Jacksonian disorders and/or involuntary tremors. For example,some embodiments are directed to treating and preventing convulsivedisorders in neonates and/or infants. Gluconate compositions have beenfound to be selectively effective in treating and/or preventingconvulsive disorders in neonates and/or infants.

Neonatal seizure has been difficult to treat compared to adult epilepsy.In one embodiment, the present invention contemplates the use of ananti-epileptic drug, gluconate, which potently suppresses neonatalepilepsy by targeting at CLC-3 Cl− channels.

Gluconic acid is a large organic anion, often used as a food or drugadditive in a salt form such as magnesium gluconate, calcium gluconate,or potassium gluconate, where gluconate was used to deliver ions.However, the functional role of gluconate itself was largely neglectedin the past. The data presented herein shows that gluconic acid itselfacts as a Cl− channel blocker and directly inhibits epileptiformactivity. Furthermore, it is shown that gluconate is particularlyeffective for suppressing neonatal seizure, and its potency is superiorto other currently available anti-epileptic drugs. Because gluconate issafe for human consumption, the compound represents a new platform forthe treatment of neonatal epilepsy.

Gluconate has been widely used as an anion in many medically used drugsbut is often labeled as inactive ingredient. In the relevant researchliterature, gluconate itself has never been directly linked to epilepsy,because the specific aims of these studies were focused on cations suchas Ca²⁺ and Mg²⁺. For example, one case study reported that an epilepticpatient was treated with Ca-gluconate and subsequently the epilepticjerks faded. Boulenguez et al., “Down-regulation of thepotassium-chloride cotransporter KCC2 contributes to spasticity afterspinal cord injury” Nature Medicine 16:302-307 (2010). The analysisattributed the ameliorative effect to Ca²⁺, without any mention of apossible role for gluconate. Other observations that magnesium gluconatemight have an anti-epileptic effect, have been suggested to be due tothe function of Mg²⁺.

The data presented herein suggest that the active ingredient in theseanti-epileptic compositions is more likely gluconate rather than thedivalent cation. Gluconate can be generated from the oxidation ofglucose, and therefore abundant in natural food product such as fruitand honey. Anastassiadis et al., “Gluconic acid production” RecentPatents On Biotechnology 1:167-180 (2007). In one embodiment, thepresent invention contemplates that gluconate has antiepileptic effectand may support certain traditional diet treatments for epilepsy.

CLC-3 channels are voltage-gated outward rectifying Cl− channels, whichbelong to the CLC super family. Previous studies have shown that CLC-3channels are essential for the regulation of endosomal acidification,presynaptic neurotransmitter accumulation, insulin secretion, and gliomacell proliferation. Hara-Chikuma et al., “ClC-3 chloride channelsfacilitate endosomal acidification and chloride accumulation” TheJournal Of Biological Chemistry 280:1241-1247 (2005); Riazanski et al.,“Presynaptic CLC-3 determines quantal size of inhibitory transmission inthe hippocampus” Nature Neuroscience 14:487-494 (2011); Deriy et al.,“The granular chloride channel ClC-3 is permissive for insulinsecretion” Cell Metabolism 10:316-323 (2009): Li et al., “Suppression ofsulfonylurea- and glucose-induced insulin secretion in vitro and in vivoin mice lacking the chloride transport protein ClC-3” Cell Metabolism10:309-315 (2009); Cuddapah et al., “Bradykinin-induced chemotaxis ofhuman gliomas requires the activation of KCa3.1 and ClC-3” The JournalOf Neuroscience: The official journal of the Society for Neuroscience33:1427-1440 (2013); and Habela et al., “ClC3 is a critical regulator ofthe cell cycle in normal and malignant glial cells” The Journal OfNeuroscience: the official journal of the Society for Neuroscience28:9205-9217 (2008).

Although it is not necessary to understand the mechanism of aninvention, it is believed that CLC-3 Cl− channels mediate a largeoutward rectifying Cl− current that is prominent in early postnatalbrains but largely disappeared in adult brains. Consequently, gluconateis a more potent anti-epileptic drug for the treatment of neonatalepilepsy. It is further believed that the activation of CLC-3 Cl−channels during epileptogenesis alters the intracellular Cl− homeostasisand makes GABA function more excitatory. Furthermore, the data presentedherein shows that the CLC-3 Cl− channels may play a bigger role inneonatal epilepsy than previously thought Cl− transporters. In fact, Cl−channels and Cl− transporters may play different roles in controllingintracellular Cl− homeostasis due to their differences in voltagedependence. For example, in resting a condition voltage-sensitive CLC-3Cl− channels are inactive, and therefore Cl− transporters such as NKCC1is the major player that maintains intracellular Cl− homeostasis indeveloping neurons. Ben-Ari, Y., “Excitatory actions of gaba duringdevelopment: the nature of the nurture” Nature Reviews. Neuroscience3:728-739 (2002); Kaila et al., “Cation-chloride cotransporters inneuronal development, plasticity and disease” Nature Reviews.Neuroscience 15:637-654 (2014); and Blaesse et al., “Cation-chloridecotransporters and neuronal function” Neuron 61:820-838 (2009).

During epileptogenesis, however, CLC-3 Cl− channels are activated andthe large Cl− influx significantly increases [Cl−]_(i), resulting inenhanced excitatory GABAergic transmission and exacerbated epilepticactivity. In some embodiments, the present invention contemplates thatblocking CLC-3 channels with gluconate disrupts this positive loop andinhibit neonatal epilepsy in the developing brains. Moreover, CLC-3knockout mice do not have large outward rectifying Cl− current, andconsequently the recurrent burst activity is significantly diminished.

By comparing gluconate with other potential anti-epileptic drugs (AEDs),the data presented herein shows that gluconate is more potent thanphenobarbital or bumetanide in suppressing neonatal epilepsy. Gluconateacts differently from bumetanide, which blocks NKCC1 and affects EGABAunder resting condition, whereas gluconate does not affect normal EGABAunder physiologic condition, but blocks CLC-3 channel activation duringepileptogenesis.

Since gluconate is a natural organic acid and already used as a food anddrug additive with minimal side effects. In one embodiment, the presentinvention contemplates that gluconate compositions, in the absence of adivalent cation, are therapeutic drugs that can be used for thetreatment of neonatal epilepsy that is resistant to many currentanticonvulsant drugs.

I. Convulsive Disorders

The term convulsion is often used interchangeably with seizure.Convulsions occur when a person's body shakes rapidly anduncontrollably. During convulsions, the person's muscles contract andrelax repeatedly. There are many different types of seizures. Some havemild symptoms without shaking. Some seizures only cause a person to havestaring spells. These may go unnoticed. Specific symptoms of convulsionsmay depend on what part of the brain is involved. Symptoms usually occursuddenly and may include, but are not limited to: brief blackoutfollowed by a period of confusion (the person cannot remember for ashort time); changes in behavior such as picking at one's clothing;drooling or frothing at the mouth; eye movements; grunting and snorting;loss of bladder or bowel control; mood changes such as sudden anger,unexplainable fear, panic, joy, or laughter; shaking of the entire body;sudden falling, tasting a bitter or metallic flavor; teeth clenching;temporary stop in breathing, and/or uncontrollable muscle spasms withtwitching and jerking limbs. Symptoms may stop after a few seconds orminutes, or continue for up to 15 minutes but they rarely continuelonger.

Epileptic seizures are often caused by overexcitation of the braincircuits, which can be inhibited by boosting GABA_(A) receptors(GABA_(A)-Rs), a major inhibitory receptor family in the adult brain.Therefore, anti-epileptic drugs (AEDs) are often developed to increaseGABAA-R function, such as benzodiazepine and barbiturate drugs that aregenerally targeted towards adult patients. Bialer et al., “Key factorsin the discovery and development of new antiepileptic drugs. Naturereviews. Drug discovery 9, 68-82 (2010). However, while GABA_(A)-Rs aremostly inhibitory in the adult brain, GABA_(A)-Rs are mostly excitatoryin the developing brain of neonates and/or infants. Chen et al.,“Excitatory actions of GABA in developing rat hypothalamic neurones” TheJournal Of Physiology 494(Pt 2):451-464 (1996); and Ben-Ari Y.,“Excitatory actions of gaba during development: the nature of thenurture” Nature Reviews Neuroscience 3:728-739 (2002). The excitatoryGABAergic transmission in the developing brain also explains why GABAagonists are often ineffective in controlling neonatal seizures, andsometimes can even exacerbate neonatal seizure activity. Farwell et al.,“Phenobarbital for febrile seizures—effects on intelligence and onseizure recurrence” The New England Journal Of Medicine 322:364-369(1990). Therefore, some AEDs can potentially exacerbate neonatalseizures by enhancing excitatory GABA_(A)-R function in the developingbrain.

Classically, the excitatory function of GABA_(A)-Rs in developingneurons has been attributed to the regulation by chloride ion (Cl⁻)transporters NKCC1 and KCC2. Ben-Ari et al., “GABA: a pioneertransmitter that excites immature neurons and generates primitiveoscillations” Physiol Rev 87:1215-1284 (2007); Kahle et al., “Roles ofthe cation-chloride cotransporters in neurological disease” Nat ClinPract Neurol 4:490-503 (2008); and Kaila et al., “Cation-chloridecotransporters in neuronal development, plasticity and disease” NatureReviews. Neuroscience 15:637-654 (2014). A previous study found thatNKCC1 might facilitate neonatal seizures in rodent animals. Dzhala etal., “NKCC1 transporter facilitates seizures in the developing brain”Nature Medicine 11:1205-1213 (2005). However, a recent clinical trial ininfant babies found severe side effects of NKCC1 blocker bumetanide andvery limited effect in treating neonatal seizure. Pressler et al.,“Bumetanide for the treatment of seizures in newborn babies with hypoxicischaemic encephalopathy (NEMO): an open-label, dose finding, andfeasibility phase 1/2 trial” Lancet Neurol 14:469-477 (2015). Becauseexcitatory GABAergic transmission plays a fundamental role in manyneural developmental processes, blocking NKCC1 may significantly alterGABA function under physiological condition and raise a potential riskof disrupting normal brain development. Ben-Ari, Y., “Excitatory actionsof gaba during development: the nature of the nurture” Nature reviews.Neuroscience 3:728-739 (2002); Kaila et al., “Cation-chloridecotransporters in neuronal development, plasticity and disease” Naturereviews. Neuroscience 15:637-654 (2014); Wang et al., “Blocking earlyGABA depolarization with bumetanide results in permanent alterations incortical circuits and sensorimotor gating deficits” Cerebral cortex21:574-587 (2011); and Deidda et al., “Early depolarizing GABA controlscritical-period plasticity in the rat visual cortex” Nature neuroscience18:87-96 (2015). More recent studies suggest that factors other than Cl⁻co-transporters may also contribute to Cl⁻ homeostasis, such asimpermeant anions, voltage-gated inward rectifying chloride channelCLC-2, and voltage-gated outward rectifying chloride channel CLC-3.Glykys et al., “Local impermeant anions establish the neuronal chlorideconcentration” Science 343:670-675 (2014); Foldy et al., “Regulation offast-spiking basket cell synapses by the chloride channel ClC-2” Natureneuroscience 13:1047-1049 (2010); Rinke et al., “ClC-2 voltage-gatedchannels constitute part of the background conductance and assistchloride extrusion” The Journal of neuroscience: the official journal ofthe Society for Neuroscience 30:4776-4786 (2010); Kawasaki et al.,“Cloning and expression of a protein kinase Cl− regulated chloridechannel abundantly expressed in rat brain neuronal cells” Neuron12:597-604 (1994); and Wang et al., “CLC-3 channels modulate excitatorysynaptic transmission in hippocampal neurons” Neuron 52:321-333 (2006).

Recent studies suggest that Cl⁻ channels, such as CLC-3 channels, mayalso contribute to the changes of intracellular Cl⁻ concentration([Cl⁻]_(i)). Zhou et al., “Regulation of intracellular Cl− concentrationthrough volume-regulated ClC-3 chloride channels in A10 vascular smoothmuscle cells” The Journal Of Biological Chemistry 280:7301-7308 (2005).CLC-3 channels belong to a subfamily of voltage-dependent outwardrectifying Cl⁻ channels. Jentsch T. J., “Chloride and theendosomal-lysosomal pathway: emerging roles of CLC chloridetransporters” The Journal of Physiology 578:633-640 (2007); Graves etal., “The Cl−/H+ antiporter ClC-7 is the primary chloride permeationpathway in lysosomes” Nature 453:788-792 (2008); Picollo et al.,“Chloride/proton antiporter activity of mammalian CLC proteins ClC-4 andClC-5” Nature 436:420-423 (2005); Scheel et al., “Voltage-dependentelectrogenic chloride/proton exchange by endosomal CLC proteins” Nature436:424-427 (2005); and Hara-Chikuma et al., “ClC-3 chloride channelsfacilitate endosomal acidification and chloride accumulation” TheJournal Of Biological Chemistry 280:1241-1247 (2005). CLC-3 channels areubiquitously expressed throughout the brain, with high expression levelin the hippocampus and cerebellum. Duran et al., “Chloride channels:often enigmatic, rarely predictable” Annual Review Of Physiology72:95-121 (2010); Verkman et al., “Chloride channels as drug targets”Nature Reviews. Drug discovery 8:153-171 (2009); and Kawasaki et al.,“Cloning and expression of a protein kinase Cl− regulated chloridechannel abundantly expressed in rat brain neuronal cells” Neuron12:597-604 (1994). For example, CLC-3 knockout mice show selectivepostnatal neurodegeneration in the hippocampus. Stobrawa et al.,“Disruption of ClC-3, a chloride channel expressed on synaptic vesicles,leads to a loss of the hippocampus” Neuron 29:185-196 (2001); andDickerson et al., “Altered GABAergic function accompanies hippocampaldegeneration in mice lacking ClC-3 voltage-gated chloride channels”Brain Research 958:227-250 (2002). However, the precise role ofvoltage-dependent Cl− channels during epileptogenesis is largelyunknown.

Neonatal seizure has been difficult to treat as compared to adultepilepsy. Neonatal seizure is different from adult seizure, and manyantiepileptic drugs (AEDs) that are effective in adults often fail totreat neonatal seizure. For example, the data presented herein shows alarge voltage-dependent outward rectifying Cl− current mediated by CLC-3Cl− channels that is present in early developing brains but not in anadult brain. Although it is not necessary to understand the mechanism ofan invention, it is believed that gluconate, a naturally-occurringorganic acid, potently inhibits neonatal seizure by blocking CLC-3chloride channels.

Interestingly, CLC-3 Cl− channels may be upregulated duringepileptogenesis and inhibition of CLC-3 Cl− channels by gluconateessentially abolishes neonatal seizure activity, with a significantlyimproved potency as compared to other AEDs. It is believed that thisresult may be explained by the observation that CLC-3 knockout mice haveno outward rectifying Cl− current in developing brains and show reducedrecurrent epileptiform activity. Although it is not necessary tounderstand the mechanism of an invention, it is believed that activationof CLC-3 Cl− channels during epileptogenesis significantly altersintracellular Cl− homeostasis and enhances GABA excitatory function, aneffect also observed with other Cl− modulators. The data presentedherein identifies gluconate as a potent anti-epileptic drug to treatneonatal seizure through inhibiting CLC-3 Cl− channels. In someembodiments described herein, an anti-epileptic drug, gluconic acid, isidentified that is particularly potent in suppressing neonatal seizure.

II. Divalent Cation-Based Convulsive Therapies

The administration of calcium gluconate, magnesium sulphate orphenobarbitone to 4-7 day old infants having convulsions associated withhypocalcemia has been reported. While all three compositions appeared toreduce the rate of convulsions, intra-treatment comparisons showed thatmagnesium sulfate was more effective than either calcium gluconate orphenobarbitone. The reference, however, does not provide any disclosureas to the effectiveness of a gluconate composition in an infant that isnot in need of a cation-based therapy. Turner, T., “Comparisons ofphenobarbitone, magnesium sulphate, and calcium gluconate in treatmentof neonatal hypocalcaemic convulsions” Paediatric Research SocietyAbstracts, pg 244.

The administration of calcium gluconate to an infant of less than twentydays has been reported for the management of convulsions. Subsequent tothis treatment, the left leg underwent swelling, inflammation,extraosseus calcification and the serum calcium levels were determinedto be within normal limits. These symptoms were relieved followingcessation of the calcium gluconate treatment with a diagnosis ofiatrogenic calcinosis cutis. The reference, however, does not provideany disclosure as to the effectiveness of a gluconate composition in aninfant that is not in need of a divalent cation-based therapy, such ascalcium. Arora et al., “Iatrogenic calcinosis cutis—a rare differentialdiagnosis of soft-tissue infection in a neonate: A case report” Journalof Orthopaedic Surgery 13(2):195-198 (2005).

The administration of botanical extracts for treating seizure disordersand/or epilepsy has been reported. For example, botanical extracts havebeen combined with various formulations of gluconate, for example,potassium gluconate, zinc gluconate and/or copper gluconate and screenedfor anticonvulsant activity using a known mouse electroshock clonusmodel. Further, a commercially available product, NutriiVeda™, wasadministered to human patients ranging in age from between four andone-half (4.5) to thirty seven (37) years old to reduce seizures and/orepilepsy. It should be noted, however, a detailed compositional analysisof NutriiVeda™ was not disclosed, therefore, the exact amount ofgluconate, and its relationship to divalent cations, in this product isnot known. The reference, however, does not provide any disclosure as tothe effectiveness of a gluconate composition in an infant that is not inneed of a divalent cation-based therapy, such as calcium or zinc. GengL., “Methods For Treating Neurological Disorders Using NutrientCompositions” U.S. Pat. No. 8,962,042 (herein incorporated byreference).

The case of a neonate was presented who had early onset seizureassociated with hypocalcemia, hyperphosphatemia, and raised parathyroidhormone. The infant did not have any stigmata ofpseudohypoparathyroidism. The hypocalcemia was initially resistant tocalcium therapy, but responded to vitamin D analog therapy. Thediagnosis of ‘neonatal pseudohypoparathyroidism’ was entertained; theinfant remained stable and seizure-free with normal serum biochemistryduring 3 and 8 months of follow-up. Narang et al., “Neonatalpseudohypoparathyroidism” Indian J Pediatr. 73(1):97-98 (2006); andManzar et al., “Transient pseudohypoparathyroidism and neonatal seizure”J Trop Pediatr. 47(2):113-114 (2001).

The failure of calcium-based convulsion therapy has been reportedregarding a patient that developed generalized tonic-clonic seizureswhen she was 9 years old and these were associated with hypocalcemia.Despite treatment with calcium, seizures persisted and the patientrequired antiepileptic medications. She was eventually controlled withoxcarbazepine. An MRI of the head was normal. An EEG showed independentspike and wave discharges emanating from the left temporal and rightfrontal region. The presence of focal findings on EEG, the lack ofcomplete response to calcium therapy, and the need for antiepilepticdrug therapy indicate that some of these patients may be inherentlypredisposed to developing epilepsy. Gonzalez et al., “Seizures and EEGfindings in an adult patient with DiGeorge syndrome: a case report andreview of the literature” Seizure 18(9):648-651 (2009).

Hypocalcemia is a relatively uncommon but reversible cause of leftventricular dysfunction in infants and children. A 30-day-old boy withidiopathic hypocalcemia presented with congestive heart failure andconvulsive seizures. He had no evidence of underlying cardiac disease.The cardiac failure responded to calcium therapy. It is suggested thathypocalcemia should be considered as a possible cause of leftventricular dysfunction in infants. Karademir et al., “Left ventriculardysfunction due to hypocalcemia in a neonate” JPN Heart J. 34(3):355-359(1993).

Such reports as above teach one of skill in the art that the purpose ofadministering a divalent-cation gluconate therapy is to provide thedivalent cation (e.g., calcium, magnesium, zinc etc.) as the effectiveingredient to manage the convulsive disorder. For example, the conditionof“hypocalcemia” is described as a pre-existing condition for theconvulsive condition. On the other hand, calcium excess resulted inserious side effects during convulsive treatments experienced subsequentto the treatment of calcium gluconate. As such, these observations teachthat the purpose of administering a divalent-cation gluconate complex tothe neonate is because it was believed that divalent-cationsupplementation was required for convulsion management, and gluconatewas merely a suitable counter ion. Consequently, the present inventiondemonstrates that divalent-cation gluconate compositions aresurprisingly more effective in treating convulsive conditions.

III. Gluconate-Based Convulsive Therapies

In one embodiment, the present invention contemplates thatvoltage-dependent CLC-3 Cl− channels may play a role in controllingintracellular Cl− concentration ([Cl−]_(i)), particularly duringneonatal epilepsy. For example, CLC-3 channels may mediate a largevoltage-dependent outward rectifying Cl− currents in neonatal but notadult brains that can be inhibited by gluconic acid. For example, thedata show that gluconate potently suppressed neonatal epilepticactivity, with a less effect in adult animals. Moreover, CLC-3 knockoutmice showed an absence of voltage-dependent outward rectifying Cl−currents in neonatal brains and reduced recurrent epileptic activity.EEG recordings also confirmed that gluconate was more effective ininhibiting seizure burst activity in neonatal animals, but lesseffective in adult animals. Finally, CLC-3 channel activation observedduring neonatal epilepsy significantly increased [Cl−]_(i), whileblocking CLC-3 channels using gluconate inhibited [Cl−]; accumulation.In one embodiment, the present invention contemplates that gluconate (anatural organic chemical) is an effective CLC-3 Cl− channel blocker andmay be effective as an anticonvulsant drug to treat neonatal epilepsy.

Gluconic acid is a large organic anion, often used as a food or drugadditive in a salt form such as magnesium gluconate, calcium gluconate,or potassium gluconate, where gluconate was used to help deliveringcalcium or magnesium or potassium. However, the functional role ofgluconate itself was largely neglected in the past. The data presentedherein demonstrate that gluconic acid itself acts as a Cl− channelblocker and directly inhibits epileptiform activity. In someembodiments, the present invention provides that gluconate isparticularly effective for suppressing neonatal seizure thereby providea selective therapy for the treatment of neonatal epilepsy.

Gluconate is an alkanoyl organic compound having the followingstructure:

It is noted that the various hydroxyl groups and carboxylic acid groupprovide reactive species capable of functionalization and/orderivitization. Such reactive groups may be substituted with moietiesincluding, but not limited to, a substituted or unsubstituted aryl orheteroaryls, an unsubstituted or substituted C1-C6-alkyl group, asubstituted or unsubstituted 5-6-membered saturated or unsaturated fusedring, a substituted or unsubstituted 5-6-membered saturated ornon-saturated ring, natural amino acid residues or synthetic amino acidresidues, trihalomethyl, substituted or unsubstituted C1-C6-alkoxy, NH₂,SH, thioalkyl, aminoacyl, aminocarbonyl, substituted or unsubstitutedC1-C6-alkoxycarbonyl, aryl, heteroaryl, substituted or unsubstituted4-8-membered cyclic alkyl, optionally containing 1-3 heteroatoms,carboxyl, cyano, halogen, hydroxy, nitro, acetoxy, aminoacyl, sulfoxy,sulfonyl, C1-C6-thioalkoxy, C1-C6-aliphatic alkyl, substituted orunsubstituted saturated cyclic C4-C8-alkyl optionally containing 1-3heteroatoms and optionally fused with an aryl or an heteroaryl; asubstituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, whereby said aryl or heteroaryl groups are optionallysubstituted with substituted or unsubstituted C1-C6-alkyl, liketrihalomethyl, substituted or unsubstituted C1-C6-alkoxy, substituted orunsubstituted C2-C6-alkenyl, substituted or unsubstituted C2-C6-alkynyl,amino, aminoacyl, aminocarbonyl, substituted or unsubstitutedC1-C6-alkoxycarbonyl, aryl, carboxyl, cyano, halogen, hydroxy, nitro,acetoxy, aminoacyl, sulfoxy, sulfonyl, C1-C6-thioalkoxy; or R5 and R6taken together could form a substituted or unsubstituted 4-8-memberedsaturated cyclic alkyl or heteroalkyl group. It is expected that atleast one or more of these contemplated gluconate derivatives haveanticonvulsant effect that is superior to that of traditionally useddivalent cation therapies.

Gluconate has been widely used as an anion in many medically used drugsand is usually labeled as an inactive ingredient. Those skilled in theart reporting anticonvulsive compounds having gluconate as a counterion, have never suggested that gluconate is linked to any anti-epilepticeffect. Instead, as discussed above, previous studies primarily focus ondivalent cations such as Ca²⁺ and Mg²⁺, as mediating the anticonvulsiveeffect. For example, one case study reported that an epileptic patientwas treated with Ca-gluconate and epileptic jerks faded. Boulenguez etal., “Down-regulation of the potassium-chloride cotransporter KCC2contributes to spasticity after spinal cord injury” Nature Medicine16:302-307 (2010). The authors attributed this effect to Ca²⁺, totallyignoring the question as to whether gluconate may have played any role.Similarly, it is generally accepted in the art that magnesium-relatedanti-convulsive products, such as magnesium gluconate, might have ananti-epileptic effect, but again, it is believed that theanti-convulsive effect is due to the function of Mg²⁺. In contrast tothese generally held beliefs, the data presented herein demonstrate thatgluconate may be just as, or more, effective than a divalent cationalone.

Gluconate can be generated from the oxidation of glucose, and istherefore abundantly found in natural food product, such as fruit andhoney. Anastassiadis et al., “Gluconic acid production” Recent PatentsOn Biotechnology 1:167-180 (2007). Consequently, a dietary source ofgluconate may also contribute to an anti-epileptic therapy. Sincegluconate is a natural organic acid and already used as food and drugadditive with minimal side-effects, gluconate derivatives could resultin a new generation of therapeutic drugs for the treatment of neonatalepilepsy.

In one embodiment, the present invention contemplates that gluconic acid(e.g., gluconate) potently inhibited epileptiform burst activity inneuronal cultures as well as in hippocampal slices. Further, fieldpotential recordings in hippocampal slices revealed that sodiumgluconate potently suppressed neonatal epileptic activity, with lesseffect in older animals. Electroencephalographic (EEG) recordingsconfirmed that sodium gluconate was more effective in inhibitingepileptic seizures in neonatal animals, but less effective in adultanimals. Whole-cell patch clamp recordings demonstrated that sodiumgluconate significantly inhibited the voltage-dependent Cl⁻ currents inhippocampal pyramidal neurons, which are mainly mediated by CLC-3channels. The data presented herein identify a natural organic chemical,gluconic acid and derivatives thereof, as previously unknown, andeffective, anticonvulsant drugs for neonatal epilepsy.

A. Inhibition of Epileptiform Activity

The functional role of Cl⁻ was investigated during epileptogenesis byreplacing extracellular Cl⁻ (i.e., for example, 137 mM Cl⁻) in the bathsolution with large anions such as gluconic acid. Surprisingly, aconcentration of between approximately 5-20 mM sodium gluconic acid(NaGcA) in the bath solution, inhibited spontaneous epileptiformactivity in cultured cortical neurons. See, FIG. 1A. Such potentinhibition of epileptiform activity by low concentration of NaGcA couldnot be explained purely by Cl⁻ concentration change, because there wasstill >117 mM Cl⁻ in the bath solution. To determine whether NaGcA mightdirectly inhibit epileptiform activity, cyclothiazide (CTZ) was used toelicit robust epileptiform burst activity in neuronal cultures. Qi etal., “Cyclothiazide induces robust epileptiform activity in rathippocampal neurons both in vitro and in vivo” The Journal Of Physiology571:605-618 (2006). The data demonstrated that CTZ-induced epileptiformactivity was completely blocked by 10 mM NaGcA, and the inhibition wasdose-dependent See, FIG. 1B and FIG. 1C, respectively. The effect ofNaGcA was reversible, because the burst activity was restored to thecontrol level after washing out NaGcA. See, FIG. 1A and FIG. 1C. Thesedata indicate that NaGcA exerts a potent inhibitory effect onepileptiform burst activity in cultured neurons.

To ensure that such inhibitory effect of NaGcA was not limited to theCTZ epilepsy model, NaGcA was further tested in two additional epilepticmodels. Firstly, cortical neurons were treated with 1 μM kainic acid(KA), a potent neurotoxin, for 2 hrs to induce epileptic burst activity.Whole-cell patch-clamp recordings showed that 10 mM NaGcA greatlysuppressed the epileptiform burst activity induced by KA. See, FIG. 1Dand FIG. 1E. Secondly, cortical neurons were incubated in 50 μM4-aminopyridine (4-AP) for 2 hrs, which also induced long-lastingepileptiform burst activity. Similarly, application of 10 mM NaGcAcompletely blocked the 4-AP-induced epileptiform activity. See, FIG. 1Fand FIG. 1G. Together, these results demonstrate that NaGcA is a potentand general inhibitor for epileptiform activity in cultured neurons.

Whether NaGcA can inhibit the induction of epileptic activity was thendetermined. For this purpose, a combination of NaGcA and CTZ was addedduring the induction period. Neurons treated by CTZ alone showed robustepileptiform activity; however, neurons treated with 10 μM CTZ togetherwith 10 mM NaGcA showed a great reduction of epileptiform activity. See,FIG. 1H, cf, top portion to bottom portion. Almost 90% of controlneurons showed epileptiform activity after CTZ-treatment (e.g.,approximately 19 out of 22 neurons), whereas only 18% (e.g.,approximately 3 out of 17 neurons, p<0.001, χ2 test) of neuronsco-treated with 10 mM NaGcA showed epileptiform activity. See, FIG.1I(a). The average frequency and duration of epileptiform bursts werealso significantly reduced in neurons co-treated with NaGcA. See, FIG.1I(b) and FIG. 1I(c). Together, these data demonstrate that both acuteand chronic NaGcA application can suppress epileptiform activity incultured neurons.

B. Kainic Acid-Induced Cell Death Protection

Neuronal death is the serious side-effect of epilepsy. Sagar et al.,“Hippocampal neuron loss in temporal lobe epilepsy: correlation withearly childhood convulsions” Annals of Neurology 22:334-340 (1987); andSass et al., “Verbal memory impairment resulting from hippocampal neuronloss among epileptic patients with structural lesions” Neurology45:2154-2158 (1995). The data presented herein demonstrate thatgluconate not only has anti-epileptic effect, but also exerts neuralprotective effect against KA-induced neuronal death.

Epileptic activity may induce cell death both in epileptic patients andanimal models. Chen et al., “Differential roles of NR2A- andNR2B-containing NMDA receptors in activity-dependent brain-derivedneurotrophic factor gene regulation and limbic epileptogenesis” TheJournal Of Neuroscience: the official journal of the Society forNeuroscience 27:542-552 (2007); and Naseer et al., “Maternal epilepticseizure induced by pentylenetetrazol: apoptotic neurodegeneration anddecreased GABAB1 receptor expression in prenatal rat brain” MolecularBrain 2:20 (2009). To evaluate whether the anti-epileptic effect ofNaGcA might be neuroprotective, a kainic acid (KA)-induced cell deathmodel was used in conjunction with the LIVE/DEAD® Viability/CytotoxicityAssay Kit (L3224, Life Technologies, Inc.) to analyze the neuronalsurvival rate. Wu et al., “Protective effect of resveratrol againstkainate-induced temporal lobe epilepsy in rats” Neurochemical Research34:1393-1400 (2009). In a control group, most neurons appeared to behealthy in morphology and were stained by calcein (green, live) but notethidium homodimer-1 (EthD-1, red, death). See, FIG. 1J, left. Afterexposure to pM KA for 24 hrs, most neurons were dead as shown by EthD-1staining. See, FIG. 1J, middle left). Interestingly, KA-induced neuronaldeath was abolished by co-application of 20 mM NaGcA. See, FIG. 1J,middle right. Neurons exposed to 20 mM NaGcA alone had no side effect ontheir survival rate. See, FIG. 1J, right. Quantitative data showed thatthe neuroprotective effect of NaGcA was also dose-dependent. See, FIG.1K. These experiments suggest that gluconate not only inhibitsepileptiform activity but also exerts neuroprotective effect.

C. Chloride Ion Current Inhibition

The studies presented herein demonstrate that gluconic acid blocks Cl−channels. Although it is not necessary to understand the mechanism of aninvention, it is believed that gluconic acid blocks these channelsbecause it is a large organic compound with negative charge. Forexample, as a large anion, it is believed that gluconic acid may notpass through the Cl⁻ channel as easily as Cl− ion itself. Cl⁻ channelshave been linked to human epilepsy patients. Mutations in CLC-1 channelswere identified in many idiopathic epileptic patients. Chen et al.,“Novel brain expression of ClC-1 chloride channels and enrichment ofCLCN1 variants in epilepsy” Neurology 80:1078-1085 (2013). CLC-2 channelmutations have also been found in human patients but some studiessuggest that the mutations may not contribute to epilepsy. Haug et al.,“Mutations in CLCN2 encoding a voltage-gated chloride channel areassociated with idiopathic generalized epilepsies” Nat Genet 33:527-532(2003); Kleefuss-Lie et al., “CLCN2 variants in idiopathic generalizedepilepsy” Nat Genet 41:954-955 (2009); and Niemeyer et al., “No evidencefor a role of CLCN2 variants in idiopathic generalized epilepsy” NatGenet 42:3 (2010). CLC-3 channels are widely expressed in differentbrain regions, and hippocampus is one of the highest expression regions.Duran et al., “Chloride channels: often enigmatic, rarely predictable”Annual Review Of Physiolog 72:95-121 (2010); Verkman et al., “Chloridechannels as drug targets” Nature Reviews. Drug Discovery 8:153-171(2009); and Kawasaki et al., “Cloning and expression of a protein kinaseC-regulated chloride channel abundantly expressed in rat brain neuronalcells” Neuron 12:597-604 (1994).

Experiments were designed to answer the question as to what is/are themolecular target(s) of gluconate. Ion channel regulation was firstinvestigated where a comparison of NaGcA on sodium, potassium or calciumchannels was made. The data show that there were no significant changesin Na⁺, K⁺ and Ca²⁺ currents after NaGcA treatment. See, FIGS. 2A-2F.Although it is not necessary to understand the mechanism of aninvention, it is believed that since gluconic acid is an organic anion,it might affect neuronal anion channels.

The data presented herein show that the Cl⁻ currents in culturedcortical neurons were outwardly rectifying, and were significantlydecreased in the presence of 10 mM NaGcA. See, FIG. 2G,H; control,913±171 pA; NaGcA, 499±89 pA; n=7; P<0.007, paired t-test; recorded at+90 mV). Thus, one target of NaGcA may be a Cl⁻ channel. To solidify aclose link between Cl− channels and epileptogenesis, the effect of twoclassic Cl− channel blockers was examined;5-Nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and4,4′-Diisothiocyanato-2,2′-stilbenedisulfonic acid disodium salt (DIDS).Application of NPPB (100 μM) or DIDS (100 μM) significantly suppressedthe Cl− currents in cultured neurons. See, FIG. 2I,L. It was alsodemonstrated that both NPPB (100 μM) and DIDS (100 μM) significantlyinhibited the epileptiform activity induced by CTZ (10 μM, 24 hrs). See,FIG. 2M,N; n=9. Taken together, these results suggest that NaGcA caninhibit Cl⁻ channels, which are involved in epileptogenesis.

D. Neonatal Hippocampal Slice Epileptiform Activity Inhibition

The possibility that the above described effects of gluconate on incultured neurons were artifacts, the anti-epileptic activity of NaGcAwas examined in hippocampal slices with relatively intact neuronalcircuits. Field potentials were recorded in the CA3 pyramidal layer andinduced epileptic burst activity in Mg²⁺-free artificial cerebral spinalfluid (aCSF). After induction of stable epileptic burst activity (˜30min), 20 mM NaGcA was applied into Mg²⁺-free aCSF to test its effect onthe epileptic activity. The data show that NaGcA exerted ananti-epileptic effect in the brain slices from P6-P12 neonatal animals(FIGS. 2A and 2 c), but a modest effect in older animals around onemonth. See, FIGS. 3A and 3C cf, FIG. 3E. The power spectrum of fieldpotentials was then analyzed before, during and after NaGcAapplications. The amplitude of power was significantly reduced afterapplication of NaGcA in neonatal animals but only slightly reduced inolder animals (e.g., P26). See, FIGS. 3B and 3D cf, FIG. 3F.Quantitatively, NaGcA inhibited 60% of the average power of epilepticactivity in neonatal animals (P6-8, 59.6±4.3%, n=10 slices from 5 pups;P10-12, 62.1±3.8%, n=10 slices from 5 pups; P<0.001, paired t-test), butonly reduced 20% in older animals (P21-33, 23.8±4.1%, n=7 slices form 4pups; p<0.005, paired t-test). See, FIG. 3G. These results suggest thatNaGcA may have potent anti-epileptic activity in neonatal brains.

Whether the NaGcA effect on epileptic activity is generally applicableto other epilepsy models besides a 0 Mg2⁺ model in P6-P12 neonatalanimals was further determined. For example, a 4-AP model was use wherebath application of K+ channel blocker 4-AP (50 μM) induced a robustepileptic burst activity in the CA3 pyramidal layer. Further addition of20 mM NaGcA to a 4-AP-containing bath solution significantly reduced theepileptic activity, which was reversible after washout of NaGcA. SeeFIG. 4A. The amplitude of power also showed significant reduction in thepresence of NaGcA, and the summarized data showed an average of70.2±5.3% reduction of the power amplitude by NaGcA (n=9 slices from 4pups; P<0.001, paired t-test). See, FIG. 4B and FIG. 4C, respectively. Ahigh K+ model was also used where elevated extracellular K⁺ (+8.5 mM)evoked epileptic burst activity in the CA3 pyramidal layer. See, FIG.4D. Similarly, addition of 20 mM NaGcA in high K⁺ aCSF also blocked theepileptic burst activity and reduced the power amplitude. See, FIG.4D,E. Statistical analysis revealed that NaGcA significantly reduced theamplitude of power by 70.8±4.9% (n=5 slices from 2 pups; P<0.001, pairedt-test) in a high K model. See, FIG. 4F. In summary, these resultsdemonstrate that NaGcA can potently suppress neonatal epilepsy in avariety of epilepsy models.

E. CLC-3 Chloride Ion Channel Inhibition

As discussed above, NaGcA inhibited Cl− currents in cultured neurons.See, FIG. 2. Previous studies reported that hippocampal cultured neuronshad voltage-dependent CLC-3 Cl− channels. Wang et al., “CLC-3 channelsmodulate excitatory synaptic transmission in hippocampal neurons” Neuron52:321-333 (2006). As shown herein, gluconate has strong inhibitoryeffect on the outward rectifying Cl⁻ currents mediated by CLC-3channels. The postsynaptic Cl− influx through the CLC-3 channels canpotentiate the NMDA receptor-mediated excitatory currents, and overactivation of NMDA receptors is involved in seizure-induced neuronalcell death. Therefore, the neuroprotective effect of gluconate may bethrough an indirect suppression of NMDA receptor function.

While hippocampal formation is known to be one of the most frequentepileptogenic structures in the rodent brain, so far there is littlestudy directly investigating the relationship between CLC-3 channels andepilepsy, except a report that CLC-3 knock-out mice showed a markedresistance to PTZ-induced epilepsy. Dickerson et al., “Altered GABAergicfunction accompanies hippocampal degeneration in mice lacking ClC-3voltage-gated chloride channels” Brain Research 958:227-250 (2002).Electrophysiological studies presented herein found that CLC-3 channelsmediate the Cl⁻ currents in hippocampal CA3 pyramidal neurons, andsodium gluconate strongly inhibits CLC-3 channels. Further, the dataalso show that, after induction of epileptic activity, CLC-3 channelsare upregulated in neonatal hippocampal slices, and sodium gluconate canabolish the increased chloride currents.

Therefore, CLC-3 channels are apparently involved in neonatalepileptogenesis. Supporting these observations, CLC-3 channels have beenfound highly expressed in human glioma cells and can be blocked bygluconate. Olsen et al., “Expression of voltage-gated chloride channelsin human glioma cells” The Journal Of Neuroscience: the official journalof the Society for Neuroscience 23:5572-5582 (2003). Further, it hasbeen reported that a high incidence is apparent between glioma andepilepsy, and misregulation of Cl⁻ homeostasis is involved inglioma-associated epilepsy. Buckingham et al., “Glutamate release byprimary brain tumors induces epileptic activity” Nature Medicine17:1269-1274 (2011); and Pallud et al., “Cortical GABAergic excitationcontributes to epileptic activities around human glioma” Sci Transl Med6:244ra289 (2014). Together, these findings suggest a potential linkbetween Cl⁻ channels and neonatal epileptogenesis. As a potent inhibitorof CLC-3 channels, gluconate may be a previously unknown anti-epilepticdrug for the treatment of neonatal epilepsy.

Consistently, the data presented herein shows a voltage-dependentoutward rectification Cl⁻ current in CA3 pyramidal neurons inhippocampal slices. See, FIG. 5A, left. For example, Cl⁻ currents weredramatically reduced after application of NaGcA (20 mM). See, FIG. 5A,right. Quantitative data analysis of the I-V curves of the Cl− currentsbefore and after NaGcA. See, FIG. 5B. To directly test whether thevoltage-sensitive Cl⁻ current was mediated by CLC-3 Cl⁻ channels, CLC-3specific antibodies were introduced into the pipette solution to blockCLC-3 channels. Wang et al., “Functional effects of novel anti-ClC-3antibodies on native volume-sensitive osmolyte and anion channels incardiac and smooth muscle cells” American Journal Of Physiology. Heartand circulatory physiology 285: H1453-1463 (2003); and Duan et al.,“Functional inhibition of native volume-sensitive outwardly rectifyinganion channels in muscle cells and Xenopus oocytes by anti-ClC-3antibody” The Journal Of Physiology 531:437-444(2001).

Indeed, an outward rectified Cl⁻ current was greatly reduced after 10min dialysis of the CLC-3 antibody (1:100), but no obvious changes wereobserved in the control IgG-dialyzed neurons. See, FIG. 5C.Quantitatively, Cl⁻ current density in control condition was 36.2±1.4pA/pF at 90 mV (n=10 cells from 4 pups) and 33.5±2.3 pA/pF in the IgGgroup (n=10 cells from 3 pups), but decreased to 13.7±2.3 pA/pF in theCLC-3 antibody group (n=11 cells from 4 pups; P<0.001, one-way ANOVAwith Tukey post hoc tests). See, FIG. 5D. Thus, the Cl⁻ currents in CA3pyramidal neurons are mainly mediated by CLC-3 channels, consistent withprevious findings in CLC-3−/− hippocampal neurons.

NaGcA was also tested to determine whether the compound can specificallyinhibit CLC-3 channels. HEK293T cells were transfected with CLC-3-EGFPplasmid and the expression of CLC-3 channels was confirmed with theCLC-3 specific antibodies. See, FIG. 5E, and Matsuda et al.,“Overexpression of CLC-3 in HEK293T cells yields novel currents that arepH dependent” American Journal Of Physiolog. Cell physiolog 294:C251-262(2008). Whole-cell recordings also revealed large outward rectificationcurrents in CLC-3-transfected HEK293T cells (control, 1012±123 pA at +90mV, n=7 cells from 2 batches of cultures), but no currents were observedin EGFP-transfected control cells. See, FIG. 5F. Further, theapplication of 20 mM NaGcA significantly reduced CLC-3-mediated Cl⁻currents (NaGcA, 544±59 pA at +90 mV, n=7 cells from 2 batches; P<0.004compared to control, paired t-test), which was also reversible afterwashout of NaGcA (925±124 pA at +90 mV, n=7 cells from 2 batches; P>0.4compared to control, paired t-test). See, FIGS. 5F,G. Together, thesedata demonstrate that NaGcA is a potent inhibitor of CLC-3 Cl⁻ channels,which mediate the major outward rectification Cl⁻ currents in the CA3pyramidal neurons.

F. Epileptogeneic Upregulation of CLC-3 Channels

To determine whether CLC-3 channels are involved in epileptogenesis,CLC-3 expression level was determined before and after the induction ofepileptic activity in P8-P12 neonatal brain slices. Hippocampal sliceswere incubated in Mg²⁺-free aCSF for 1 hr to induce epileptic activityand then performed CLC-3 immunostaining to examine the CLC-3 expressionlevel. Control slices were incubated in normal aCSF for 1 hrcorrespondingly. Interestingly, the CLC-3 immunoreactivity inhippocampal CA3 pyramidal layer showed a significant increase in 0Mg²⁺-treated slices compared to the control. See, FIG. 6A,B; control,11.2±1.5 a.u., n=10 slices from 4 pups; 0 Mg²⁺, 20.4±2.8 a.u., n=11slices from 4 pups; P<0.02, Student's t-test. Such an elevatedexpression level of CLC-3 was further confirmed by Western blot. See,FIGS. 6C,D. Thus, the CLC-3 channels are upregulated in neonatalepileptic slices.

Furthermore, a patch-clamp recording directly measured the Cl⁻ currentsof CA3 pyramidal neurons in control and 0 Mg²⁺-treated slices.Consistent with immunostaining and Western blot results, whole-cellrecordings also revealed a significant increase of Cl⁻ currents in 0Mg²⁺-treated neurons. See, FIG. 6E,F; control, 31.2±1.8 pA/pF at +90 mV,n=15 cells from 3 pups; 0 Mg²⁺, 42.2±2.4 pA/pF, n=15 cells from 3 pups;P<0.001, one-way ANOVA with Tukey post hoc tests. Further, the majorityof Cl− currents after 0 Mg²⁺ treatment were significantly inhibited byNaGcA. See, FIG. 6E,F; NaGcA, 7.3±0.6 pA/pF, n=11 cells from 2 pups;P<0.001, one-way ANOVA with Tukey post hoc tests. These results suggestthat CLC-3 channels may play an role during neonatal epileptogenesis.

G. Neonatal Seizure Inhibition

To induce neonatal seizure, a commonly used KA model was used. Injectionof KA (2 mg/kg, i.p.) into P10-P12 neonatal mice elicited robustepileptic seizure bursts as revealed by in vivo EEG recordings. See,FIG. 7A,B. When NaGcA (2 g/kg, i.p.) was injected 10 min after KAinjection, the epileptic seizure activity was essentially abolished.See, FIG. 7C,D. Power analysis confirmed that the amplitude of power wassignificantly inhibited by NaGcA injection. See, FIG. 7E,F. These datashow that NaGcA is a potent in vivo anti-neonatal seizure drug.

H. Adult Seizure Inhibition

NaGcA was determined as to whether the compound can also be used totreat adult epilepsy. Preliminary data showed that the KA convulsionmodel has high mortality rate. However, a similar PTZ model resulted inreliable epileptic seizures in adult mice. See, FIG. 8A. The data showthat NaGcA prolonged the latency of epileptic seizure burst induced byPTZ, but did not suppress the epileptic burst activity as effective asseen in the neonatal animals. See, FIG. 8. Therefore, NaGcA may be aunique anti-epileptic drug that is more potent in treating neonatalseizure than that for adult epilepsy.

I. Chloride Ion Channel Blocker Whole Cell Inhibition

A related question is that other Cl⁻ channel blockers, such as NPPB andNFA, which also inhibit neonatal seizures also block Cl− ionchannel-related activity. See, FIG. 9 and FIG. 10. In one embodiment,the present invention contemplates that compounds including, but notlimited to, gluconate, NPPB, Niflumic acid, DIDS, flufenamic acid, canact similarly in inhibiting neonatal seizure. Although it is notnecessary to understand the mechanism of an invention it is believedthat neonatal seizure inhibition may occur by chloride ion channelinhibition.

IV. Glucose Oxidase Based Convulsive Therapies

In one embodiment, the present invention contemplates that glucoseoxidase can be used as an anti-convulsant drug to inhibit seizureactivity. Although it is not necessary to understand the mechanism of aninvention, it is believed that glucose oxidase converts glucose intogluconate (supra) that has been shown herein to reduce epilieptiformactivity. The data presented herein demonstrates that gluconic acid hasa strong inhibition effect on seizure activity in the developing brain,possibly by blocking CLC-3 Cl− channels. The data also shows thatglucose oxidase (GOx), an enzyme that oxidizes glucose into gluconicacid, also has anti-epileptiform activity in hippocampal slices fromneonatal mice.

Approximately 1% of the world population suffers from epilepsy. Althoughmany seizures in epilepsy patients can be partially controlled by drugs,about one-third of epileptic patients are resistant to antiepilepticdrugs. Sada et al., “Epilepsy treatment. Targeting LDH enzymes with astiripentol analog to treat epilepsy” Science 347:1362-1367 (2015); andJuge et al., “Metabolic control of vesicular glutamate transport andrelease” Neuron 68:99-112 (2010). Fortunately, several new antiepilepticdrugs were developed over the past years, showing some additionaleffects in those drug-resistant patients. Bialer et al., “Key factors inthe discovery and development of new antiepileptic drugs” NatureReviews: Drug Discovery 9:68-82 (2010). Unfortunately, there is no newdrug that is specifically developed for suppression of neonatalseizures.

For example, phenobarbital, an agonist for GABA receptors, wasdiscovered in 1912 and is the oldest but still now the first-choice totreat neonatal epilepsy but is limited to only 50% efficacy and exhibitsseveral side effects. Chamberlain et al., “Lorazepam vs diazepam forpediatric status epilepticus: a randomized clinical trial” JAMA311:1652-1660 (2014); Painter et al., “Phenobarbital compared withphenytoin for the treatment of neonatal seizures” The New EnglandJournal Of Medicine 341:485-489 (1999); and Khanna et al., “Limitationsof Current GABA Agonists in Neonatal Seizures: Toward GABA ModulationVia the Targeting of Neuronal Cl⁽⁻⁾ Transport” Front Neurol 4:78 (2013).Video-EEG studies have shown that phenobarbital suppresses EEGepileptiform activity less effectively than the clinically apparentconvulsion. The difference between clinical symptoms and electrographicrecording is named “electroclinical dissociation”, which can reach 80%in neonates after anti-convulsant treatment. Glykys et al., “Differencesin cortical versus subcortical GABAergic signaling: a candidatemechanism of electroclinical uncoupling of neonatal seizures” Neuron63:657-672 (2009).

Therefore, some studies have suggested that phenobarbital may resolvethe clinical symptoms of seizures due to sedation without correctingunderlying abnormal epileptiform activities in developing brains, whichmay lead to an overestimation of the true efficacy of phenobarbital.Boylan et al., “Phenobarbitone, neonatal seizures, and video-EEG” ArchDis Child Fetal Neonatal Ed 86:F165-170 (2002). Alternatively, thefasting and ketogenic dietary therapy has long been used to treatseizure and is still used primarily to treat refractory epilepsy inchildren. However, the mechanism for the fasting/ketogenic diet effectsare not fully understood. Zupec-Kania et al., “An overview of theketogenic diet for pediatric epilepsy” Nutr Clin Pract 23:589-596(2008).

It is generally believed that fasting, or a ketogenic dietary therapy,forces the body to burn fats rather than carbohydrates. Normally,carbohydrates (such as glucose) is the major energy substance forgenerating the body's major fuel source of adenosine triphosphate (ATP)and in particular the brain. However, when lacking carbohydrates, fat isbelieved to be converted into ketone bodies in the liver, where theketone bodies pass into the brain and replace glucose as an energysource. It has been reported that these ketone bodies may also suppressbrain epileptiform activity. Lima et al., “Neurobiochemical mechanismsof a ketogenic diet in refractory epilepsy” Clinics (Sao Paulo)69:699-705 (2014). For most patients with epilepsy, a ketogenic diet hasbeen suggested to lead to several adverse effects. Wheless J. W., “Theketogenic diet: an effective medical therapy with side effects” JournalOf Child Neurology 16:633-635 (2001): and Neal et al., “The ketogenicdiet for the treatment of childhood epilepsy: a randomised controlledtrial” Lancet Neurol 7:500-506 (2008). Therefore, it is necessary todevelop alternative therapeutic approaches to treat pediatric epilepsy.

Because the ketogenic diet apparently achieves control of epilepsy byforcibly changing the dietary components of the patient and the dataherein showing that gluconate has a strong inhibitory effect on neonatalepileptiform activity both in vitro and in vivo, one possible mechanismis that gluconate can be generated locally via oxidizing glucose byglucose oxidase. Glucose oxidase is known to be a type of enzymecommonly found in several species of fungi and insects and widely usedas a food additive. Glucose oxidase (GOx) catalyzes a reaction in whichglucose produces hydrogen peroxide and gluconolactone, whereingluconolactone may hydrolyze into gluconic acid. The data herein showsthat gluconic acid (e.g., gluconate) suppresses epileptiform activity.

To test the effects of GOx on neonatal epileptiform activity, a baselinefield potential was recorded in the CA3 in the horizontal section ofhippocampal slices (P10-12) and then GOx was added into the bath afterinduction of stable epileptiform activity by 0 Mg²⁺ for at least 20minutes. FIG. 20A. This data shows that epileptiform activity wassignificantly inhibited by 0.3 U/ml GOx and 1 U/ml GOx but not by 0.1U/ml Gox (n=8, p<0.001). FIGS. 20B-20D.

To confirm that glucose is involved in this GOx-mediated inhibition,glucose was replaced with lactate (5 mM) and pyruvate (3 mM) and thenincubated in 0 Mg^(Z) aCSF, where it was observed that GOx had no effecton the neonatal epileptiform activity. FIG. 20E.

It is generally believed that the available concentration of gluconicacid generated from glucose in an extracellular solution depends on bothenzyme activity and reaction time. Thus, increasing the reaction time inthe presence of low dose of GOx (e.g., 0.1 U/ml) might generate asufficient dose of gluconic acid to suppress epileptiform activity inthe developing brains. For example, 0.1 U/ml GOx was incubated for overone (1) hour in 0 Mg²⁺ aCSF. FIG. 2F; bubbled with 95% O₂/5% CO₂. Thesedata showed that after a one (1) hour incubation, 0.1 U/ml GOx showedstrong inhibitory effect on the epileptiform activity in the brainslices from neonatal mice. FIG. 20F; Cf. FIG. 20B. It should be notedthat these various GOx concentrations reduced the average power by64.3±8.2% (0.3 U/ml, n=9, p<0.001), 83.3±2.2% (1 U/ml, n=6, p<0.001) and81.4±2.0% (0.1 U/ml incubated 1 h, n=5, p<0.001), respectively. FIG.20G.

Besides evaluating the effects of GOx on 0 Mg²⁺ aCSF inducedepileptiform activity, it was further determined as to whether GOx (1U/ml) could inhibit neonatal epileptiform activity induced by i) highK+; or ii) 4-aminopyridine (4-AP)+0 Mg²⁺ aCSF. The data confirmed that 1U/ml GOx also showed potent inhibition on the epileptiform activityinduced by either the high K⁺ aCSF or 4-AP+0 Mg²⁺ aCSF in thehippocampal slices from developing brains (P10-12). FIGS. 21A-21C.Therefore, these data demonstrate that GOx has an activity that stronglysuppresses neonatal epileptiform activity.

V. CLC-3 Cl− Channels Mediate a Large Outward-Rectifying Current inDeveloping Neurons

Alteration of GABA function has been closely associated with epilepsy.GABA function may be determined by intracellular Cl− concentration([Cl−]_(i)) because GABAA receptors (GABAA-Rs) are believed to beligand-gated Cl− channels. For example, previous studies haveextensively investigated the role of Cl− transporters, such as KCC2 andNKCC1, in regulating [Cl−]_(i) and hence GABA functions. However, howother Cl− channels might affect [Cl−]_(i) and GABA function has not beenwell understood.

The data presented herein investigates whether voltage-dependent Cl−channels contribute to [Cl−]; homeostasis and/or regulate GABAfunctions. Previously, voltage-dependent Cl− currents were recorded thatrevealed a large outward rectifying Cl− current (3.3±0.3 nA) in CA3pyramidal neurons of hippocampal slices from neonatal mice (e.g.,P8-12). FIG. 11A, top left panel. Surprisingly, such voltage-dependentoutward rectifying Cl− current decreased significantly in adult brains(e.g., P60-62). FIGS. 11A and 11B. These data suggested a developmentalchange of Cl− channels during brain development. The Cl− currents werecorded from CA3 pyramidal neurons resembled the voltage-dependentCLC-3 Cl− currents reported previously in cultured hippocampal neurons.Wang et al., “CLC-3 channels modulate excitatory synaptic transmissionin hippocampal neurons” Neuron 52:321-333 (2006); and Huang et al.,“Calcium-activated chloride channels (CaCCs) regulate action potentialand synaptic response in hippocampal neurons” Neuron 74:179-192 (2012).

To test whether CLC-3 Cl− channels mediated the Cl− currents in neonatalCA3 neurons, CLC-3-specific antibodies were tested for blockade of CLC-3channels. Wang et al., “Functional effects of novel anti-ClC-3antibodies on native volume-sensitive osmolyte and anion channels incardiac and smooth muscle cells” American Journal Of Physiology, HeartAnd Circulatory Physiology 285:H1453-1463 (2003); and Duan et al.,“Functional inhibition of native volume-sensitive outwardly rectifyinganion channels in muscle cells and Xenopus oocytes by anti-ClC-3antibody” The Journal Of Physiology 531:437-444 (2001). Indeed, theoutward rectifying Cl− current was greatly reduced after 10 min dialysiswith the CLC-3 antibody (1:100), but no obvious change when dialyzedwith pre-absorbed control antibody. FIG. 11D.

Moreover, Cl− currents were directly examined in CLC-3 knockout mice.Huang et al., “ClC-3 deficiency protects preadipocytes against apoptosisinduced by palmitate in vitro and in type 2 diabetes mice” Apoptosis: AnInternational Journal On Programmed Cell Death 19:1559-1570 (2014); Liuet al., “ClC-3 deficiency prevents apoptosis induced by angiotensin IIin endothelial progenitor cells via inhibition of NADPH oxidase”Apoptosis: An International Journal On Programmed Cell Death18:1262-1273 (2013); and Zheng et al., “Deficiency of volume-regulatedClC-3 chloride channel attenuates cerebrovascular remodeling inDOCA-salt hypertension” Cardiovascular Research 100:134-142 (2013).Immunostaining confirmed the absence of CLC-3 signal in the CA3 regionof CLC-3 KO mice. FIG. 11E. Accordingly, an outward rectifying Cl−current was also largely absent in the CA3 neurons of CLC-3 KO mice (WT,33.1±1.3 pA/pF, n=10; CLC-3 KO, 9.0±1.6 pA/pF, n=6). FIGS. 11F and 11G.Together, these results indicate that CLC-3 Cl− channels mediate a largeoutward rectifying Cl− current in the neonatal brain, but such Cl−current significantly diminished in the adult brain.

Further testing found sodium gluconic acid (NaGcA) as a potent inhibitorto block the Cl− currents in neonatal brains. FIG. 11A-C; greentracings. In the adult brains or in CLC-3 KO mice, NaGcA had no effecton the Cl− currents. FIGS. 11A, 11B and 11G. These data suggest thatNaGcA may be an inhibitor for CLC-3 channels. Olsen et al., “Expressionof voltage-gated chloride channels in human glioma cells” The Journal OfNeuroscience: The Official Journal Of The Society For Neuroscience23:5572-5582 (2003).

To directly test this idea, CLC-3 channels were overexpressed in HEK293Tcells and expression was confirmed with CLC-3 specific antibody binding.FIG. 11H and Matsuda et al., “Overexpression of CLC-3 in HEK293T cellsyields novel currents that are pH dependent” American Journal OfPhysiology. Cell Physiology 294:C251-262 (2008). Whole-cell recordingsrevealed large outward rectifying Cl− currents in CLC-3-transfectedHEK293T cells (1012±123 pA, n=7), but not in EGFP-transfected controlcells. FIG. 11I. Furthermore, application of 20 mM NaGcA significantlyreduced the CLC-3 channel-mediated Cl− currents. FIGS. 11I and 11J.Thus, the present data identifies NaGcA as a potent inhibitor of CLC-3Cl− channels.

It was also found that gluconate can inhibit Cl− currents in thepresence or absence of a divalent cation (e.g., calcium or magnesium).The data presented herein compares the efficacy of Cl− currentinhibition between sodium gluconate, magnesium gluconate and gluconicacid. The data demonstrate that equivalent Cl− current inhibition isobserved with all three counterions. FIGS. 17A-E. Therefore, these datashow that it is gluconic acid, not the cation it carries, is theeffective component. Previous studies using gluconate salt oftenattribute the clinical effect to the cations it carries, and gluconicacid was thought as only food/drug additive, not the major effector. Thedata show that the art was incorrect in this interpretation, and morespecifically, that a divalent cation is not a required component forgluconate to have anti-epileptic efficacy.

VI. CLC-3 Channels And Neonatal Epilepsy

To understand the functional role of CLC-3 channels in neonatalepilepsy, CLC-3 channel expression levels were determined afterinduction of epileptiform activity by treating neonatal brain sliceswith 0 Mg²⁺ artificial cerebral spinal fluid (aCSF).

Mg2+-free aCSF induced epileptic activity significantly upregulatedCLC-3 channel expression in neonatal brain slices (e.g., P8-P12). FIGS.12A and 12B; control: 11.2±1.5 artificial units (n=10); 0 Mg²⁺: 20.4±2.8artificial units (n=11); P<0.02, Student's t-test. This upregulation ofCLC-3 channel expression subsequent to epileptiform activity was furtherconfirmed by Western blot. FIGS. 12C and 12D. Moreover, patch-clamprecordings also revealed a significant increase of outward rectifyingCl− currents after the induction of epileptiform activity, andapplication of NaGcA significantly blocked the Cl− currents. FIGS. 12Eand 12F; control: 31.2±1.8 pA/pF (n=15); 0 Mg: 42.2±2.4 pA/pF (n=15);NaGcA: 7.3±0.6 pA/pF (n=11); P<0.001, one-way ANOVA with Tukey post hoctests. These results suggest that CLC-3 channels may play a role duringneonatal epilepsy.

To directly test this hypothesis, CLC-3 channels were inhibited withNaGcA and examined for effects upon epileptic activities. Remarkably,the data showed that NaGcA exerted a potent anti-epileptic effect inbrain slices from young postnatal animals (P6-12). FIGS. 12G, 12H, 12Iand 12J. However, the anti-epileptic effect of NaGcA became much smallerin older animals, for example those around one month of age (e.g. P26).FIGS. 12K and 12L. Quantitatively, NaGcA inhibited 60% of the averagepower of epileptiform activity in early postnatal animals (P6-8:59.6±4.3% (n=10); P10-12: 62.1±3.8% (n=10); P<0.001, paired t-test).However, in older animals power was reduced by only 20% after weaning(P21-33: 23.8±4.1% (n=7); p<0.005, paired t-test). FIG. 12M. Such adramatic difference in NaGcA inhibition of epileptic activity betweenneonates and adult animals is consistent with the relative inhibition ofNaGcA on CLC-3 Cl− currents in different ages of animals, suggestingthat CLC-3 channels may play a role in neonatal epilepsy.

To further investigate the functional role of CLC-3 channels inepileptiform activity, epileptiform burst activity could be induced byMg²⁺-free aCSF in CLC-3 KO mice hippocampal slices, but for a muchshorter timeperiod when in comparison to the WT mice. Interestingly,when hippocampal slices from WT mice were pre-treated with NaGcA toblock CLC-3 channels, the epileptiform activity also did not last verylong, mimicking what was observed in CLC-3 KO mice. FIGS. 12N and 12O.Quantified data showed that when CLC-3 channels were either inhibited orknocked out, the burst latency was significantly prolonged, and thetotal burst activity significantly reduced. FIG. 12P. Together, thesedata suggest that CLC-3 Cl− channels may play a role duringepileptogenesis in early developing brains.

Whether the NaGcA effect on neonatal epileptic activity can be generallyapplicable to other epilepsy models beyond the 0 Mg2+ model was alsoexamined. For example, the K+ channel blocker 4-AP (50 μM) was addedinto the bath solution to induce robust epileptic burst activity inimmersed hippocampal slices. FIG. 13A. Addition of 20 mM NaGcA to the4-AP solution significantly reduced the epileptic activity, which wasreversible after washout of NaGcA. FIGS. 13A, 13B and 13C. 70.2±5.3%reduction of the power amplitude by NaGcA (n=9). (P<0.001, pairedt-test). Elevated extracellular K+ (8.5 mM) was also used to evokeepileptic burst activity. FIG. 13D. Similarly, addition of 20 mM NaGcAin high K+ aCSF also dramatically reduced the epileptic burst activity.FIGS. 13D, 13E and 13F. NaGcA reduced the power amplitude by 70.8±4.9%(n=5). (P<0.001, paired t-test). In summary, these results demonstratethat NaGcA can potently suppress neonatal epilepsy in a variety ofepilepsy models.

VII. Gluconate Inhibits Neonatal Epilepsy

In one embodiment, the present invention contemplates a methodcomprising treating neonatal epilepsy with a gluconate compound. In oneembodiment, the gluconate compound is administered systemically.

For example, neonatal rats were administered gluconate to determine theeffect on in vivo epileptic seizures in neonatal and adult animals. Toinduce seizures, the neurotoxin kainic acid (KA) (2 mg/kg, i.p.) wasinjected into neonatal rats (P10-12) to elicit robust seizure activitiesas revealed by in vivo EEG recordings. FIGS. 14A and 14B; Dzhala et al.,“NKCC1 transporter facilitates seizures in the developing brain” NatureMedicine 11:1205-1213 (2005). Furthermore, when NaGcA (2 g/kg, i.p.) wasinjected 10 min after KA injection, the epileptic seizure activity wasessentially abolished in neonatal animals. FIGS. 14C and 14D.

The anti-epileptic effect of gluconate was also compared with previouslyreported anti-convulsant drugs such as phenobarbital and bumetanide inneonatal animals. Phenobarbital is currently the drug of first choice totreat neonatal seizures, despite only ˜50% efficacy and potentialnegative neurodevelopmental consequences. Slaughter et al.,“Pharmacological treatment of neonatal seizures: a systematic review”Journal Of Child Neurology 28:351-364 (2013). Bumetanide is a potentloop diuretic, currently under evaluation as a prospective antiepilepticdrug. Loscher et al., “Cation-chloride cotransporters NKCC1 and KCC2 aspotential targets for novel antiepileptic and antiepileptogenictreatments” Neuropharmacology 69:62-74 (2013). While both phenobarbitaland bumetanide inhibited epileptic activity to certain degree inneonatal animals, their inhibition was not as potent as gluconate. CfFIGS. 14E-14H with FIGS. 14C and 14D. Quantitatively, when the EEG powerwas calculated in the last 30 min during a 2-hr recording period afterKA injection, the relative power was reduced by 72.3% in NaGcA group,35.5% in phenobarbital group, and 54.3% in bumetanide group,respectively. FIG. 14M. Therefore, gluconate appears to be a potentanti-epileptic drug for the treatment of neonatal seizure.

The anti-epileptic effect of gluconate was also compared in adultanimals. Unlike the strong inhibition of neonatal seizure, gluconateshowed less inhibition on adult seizure activity. FIGS. 141-14L and 14N.These data are consistent with brain slice recording results. FIGS.14G-14M. In addition, neonatal animals were tested regarding the effectof NaGcA on stable epileptic activity induced by KA, and found thatNaGcA still suppressed seizure activity one hour after KA injection.FIG. 15. Therefore, these data allows the conclusion that NaGcA is aneffective drug to treat neonatal seizure.

VIII. CLC-3 Channels Regulate Cl− Homeostasis During Epileptogenesis

Although it is not necessary to understand the mechanism of aninvention, it is believed that changes in [Cl−]_(i) may represent atleast one molecular mechanism of CLC-3 channels in neonatal epilepsybecause the CLC-3 channel is a voltage-dependent outward rectifying Cl−channel. For example, the epileptic burst activity has often lasted morethan 10 s. FIG. 16A.

To investigate the effect of such long-lasting epileptiform bursts onGABA function, gramicidin-perforated whole-cell recordings wereperformed to keep the intracellular Cl− intact in neonatal animals.GABAA receptor (GABAA-R) currents induced by the receptor agonistisoguvacine (100 μM, 50 ms) before and after a membrane-depolarizingshift (40 mV for 10 s) that mimics the epileptiform burst activity.Interestingly, the GABAA-R current was significantly increased aftermembrane depolarization shift in WT, but not CLC-3 KO neurons, nor inthe presence of CLC-3 channel blocker NaGcA. FIGS. 16B and 16C. Thesedata suggest that CLC-3 channels may regulate GABA function duringepileptogenesis in neonatal animals.

To further test this idea, the GABAA-R reversal potential (EGABA), whichgoverns GABA excitatory versus inhibitory function, was directlymeasured using gramicidin-perforated whole-cell recordings in neonatalmouse brain slices (e.g., P8-P9). As expected, epileptiform activityinduced a large depolarizing shift in EGABA after treating hippocampalslices with 0 Mg²⁺ aCSF for 1 h (aCSF: −59.2±2.0 mV, n=13; 0 Mg²⁺:−48.2±1.1 mV, n=10). FIG. 16D. Treatment with bumetanide (Bum, 10 μM), aspecific blocker for NKCC at low concentrations, induced ahyperpolarizing shift in EGABA (blue line) as compared to a controlEGABA (dashed line). FIG. 6E. These results are consistent with reportsthat NKCC imports Cl− into neuronal cells. Kaila et al.,“Cation-chloride cotransporters in neuronal development, plasticity anddisease” Nature Reviews. Neuroscience 15:637-654 (2014); Dzhala et al.,“NKCC1 transporter facilitates seizures in the developing brain” NatureMedicine 11:1205-1213 (2005); and Loscher et al., “Cation-chloridecotransporters NKCC1 and KCC2 as potential targets for novelantiepileptic and antiepileptogenic treatments” Neuropharmacology69:62-74 (2013). However, in the presence of bumetanide, epileptiformactivity still caused a depolarizing shift of EGABA, suggesting that afactor other than NKCC1 is regulating EGABA during epileptogenesis. FIG.16E, yellow line; Bum, −68.0±1.7 mV (n=9); 0 Mg²⁺+Bum, −50.2±1.4 mV(n=9); (p<0.002).

Treatment with KCC2 blocker VU0240551 (10 μM) did not affect the EGABAin neonatal animals. FIG. 16F, purple line; −60.8±1.9 mV (n=6). Thesedata may possibly be explained as due to a low expression level of KCC2at this early time. Epileptic activity also elicited a positive shift inEGABA. FIG. 16F, orange line; −48.9±1.3 mV (n=8). Therefore, theepileptiform activity-induced EGABA shift is not controlled by NKCC1 orKCC2 in neonatal animals.

On the other hand, when CLC-3 Cl− channels were inhibited with NaGcA (20mM), the EGABA was not further altered when exposed to 0 Mg²⁺ aCSF. FIG.16G; 0 Mg²⁺ +NaGcA: −59.3±1.7 mV (n=6). Furthermore, in CLC-3 KO mice,the EGABA was also not changed in 0 Mg²⁺ aCSF. FIG. 16H; CLC-3 KO+0Mg²⁺: −55.6±2.1 mV (n=10). Additionally, application of NaGcA in CLC-3KO mice had no more effect on EGABA in 0 Mg²⁺ aCSF (˜56.6±2.4 mV, n=5).Inhibition of CLC-3 channels with NaGcA or knock out CLC-3, didn'tchange the normal EGABA in the normal conditions. FIG. 16G; NaGcA:−60.3±0.9 mV (n=5); CLC-3 KO: −55.4±2.9 mV (n=9).

Therefore, these results suggest that CLC-3 Cl− channels play a role incontrolling EGABA during epileptogenesis in neonatal animals. In olderanimals (e.g., P30-90), however, 0 Mg²⁺ aCSF still induced a largedepolarizing shift in EGABA in the presence of NaGcA. FIG. 16J. Thesedata are consistent with observations that the CLC-3 channel-mediatedoutward rectifying Cl− currents are substantially decreased in the adultanimals. FIG. 16A.

Besides EGABA, the effects of different drugs on GABA-induced neuronalactivity during epileptogenesis were investigated in neonatal animals(e.g., P8-9). To accurately measure GABA-induced neuronal activity,cell-attached recordings monitored spike firing elicited by localapplication of the GABAA-R agonist isoguvacine (10 μM, 30 s). Tyzio etal., “Maternal oxytocin triggers a transient inhibitory switch in GABAsignaling in the fetal brain during delivery” Science 314:1788-1792(2006). The majority of resting CA3 pyramidal neurons did not respond toisoguvacine. FIG. 16K, control, (n=32). However, after 0 Mg²⁺ treatment,68% of neurons showed spike activity upon isoguvacine application. FIG.16K, 0 Mg²⁺ (n=19). Blocking NKCC1 with bumetanide or blocking KCC2 withVU0240551 did not change the spike activity elicited by isoguvacine.FIG. 16K; 0 Mg²⁺+Bum (n=19); and 0 Mg²⁺+VU (n=21). In contrast,application of NaGcA to inhibit CLC-3 Cl− channels essentially abolishedthe spike activity induced by isoguvacine after 0 Mg²⁺ treatment. FIG.16K; 0 Mg²⁺+NaGcA (n=18). Quantified data for these observations areshown in FIG. 4L.

These data indicate that activation of CLC-3 Cl− channels in neonatalanimals enhances GABA excitatory activity during epileptogenesis, andblocking CLC-3 channels is an effective way to inhibit neonatal seizurethrough reducing excitatory GABA activity.

IX. Pharmaceutical Formulations and/or Compositions

The present invention further provides pharmaceutical compositions(e.g., comprising the compounds described above). The pharmaceuticalcompositions of the present invention may be administered in a number ofways depending upon whether local or systemic treatment is desired andupon the area to be treated. Administration may be topical (includingophthalmic and to mucous membranes including vaginal and rectaldelivery), pulmonary (e.g., by inhalation or insufflation of powders oraerosols, including by nebulizer; intratracheal, intranasal, epidermaland transdermal), oral or parenteral. Parenteral administration includesintravenous, intraarterial, subcutaneous, intraperitoneal orintramuscular injection or infusion; or intracranial, e.g., intrathecalor intraventricular, administration. In particular, an intramuscularinjection and/or intravenous injection of sodium gluconate may bedelivered in a sterile saline solution at an approximate concentrationrange of 1-100 mM gluconate. Although it is not necessary to understandthe mechanism of an invention it is believed that these concentrationranges are achievable because sodium gluconate is highly water soluble.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable.

Compositions and formulations for oral administration include powders orgranules, suspensions or solutions in water or non-aqueous media,capsules, sachets or tablets. Thickeners, flavoring agents, diluents,emulsifiers, dispersing aids or binders may be desirable.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionsthat may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, liquid syrups, soft gels, suppositories, and enemas. Thecompositions of the present invention may also be formulated assuspensions in aqueous, non-aqueous or mixed media. Aqueous suspensionsmay further contain substances that increase the viscosity of thesuspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

In one embodiment of the present invention the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product.

Agents that enhance uptake of oligonucleotides at the cellular level mayalso be added to the pharmaceutical and other compositions of thepresent invention. For example, cationic lipids, such as lipofectin(U.S. Pat. No. 5,705,188), cationic glycerol derivatives, andpolycationic molecules, such as polylysine (WO 97/30731), also enhancethe cellular uptake of oligonucleotides.

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions.Thus, for example, the compositions may contain additional, compatible,pharmaceutically-active materials such as, for example, antipruritics,astringents, local anesthetics or anti-inflammatory agents, or maycontain additional materials useful in physically formulating variousdosage forms of the compositions of the present invention, such as dyes,flavoring agents, preservatives, antioxidants, opacifiers, thickeningagents and stabilizers. However, such materials, when added, should notunduly interfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient. Theadministering physician can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC₅₀s found to be effective in in vitroand in vivo animal models or based on the examples described herein. Ingeneral, dosage is from 0.01 μg to 100 g per kg of body weight, and maybe given once or more daily, weekly, monthly or yearly. The treatingphysician can estimate repetition rates for dosing based on measuredresidence times and concentrations of the drug in bodily fluids ortissues. Following successful treatment, it may be desirable to have thesubject undergo maintenance therapy to prevent the recurrence of thedisease state, wherein the compound is administered in maintenancedoses, ranging from 0.01 μg to 100 g per kg of body weight, once or moredaily, to once every 20 years.

X. Drug Delivery Systems

The present invention contemplates several drug delivery systems thatprovide for roughly uniform distribution, have controllable rates ofrelease. A variety of different media are described below that areuseful in creating drug delivery systems. It is not intended that anyone medium or carrier is limiting to the present invention. Note thatany medium or carrier may be combined with another medium or carrier;for example, in one embodiment a polymer microparticle carrier attachedto a compound may be combined with a gel medium.

Carriers or mediums contemplated by this invention comprise a materialselected from the group comprising gelatin, collagen, cellulose esters,dextran sulfate, pentosan polysulfate, chitin, saccharides, albumin,fibrin sealants, synthetic polyvinyl pyrrolidone, polyethylene oxide,polypropylene oxide, block polymers of polyethylene oxide andpolypropylene oxide, polyethylene glycol, acrylates, acrylamides,methacrylates including, but not limited to, 2-hydroxyethylmethacrylate, poly(ortho esters), cyanoacrylates,gelatin-resorcin-aldehyde type bioadhesives, polyacrylic acid andcopolymers and block copolymers thereof.

One embodiment of the present invention contemplates a drug deliverysystem comprising therapeutic agents as described herein.

Microparticles

One embodiment of the present invention contemplates a medium comprisinga microparticle. Preferably, microparticles comprise liposomes,nanoparticles, microspheres, nanospheres, microcapsules, andnanocapsules. Preferably, some microparticles contemplated by thepresent invention comprise poly(lactide-co-glycolide), aliphaticpolyesters including, but not limited to, poly-glycolic acid andpoly-lactic acid, hyaluronic acid, modified polysaccharides, chitosan,cellulose, dextran, polyurethanes, polyacrylic acids, pseudo-poly(aminoacids), polyhydroxybutrate-related copolymers, polyanhydrides,polymethylmethacrylate, poly(ethylene oxide), lecithin andphospholipids.

Liposomes

One embodiment of the present invention contemplates liposomes capableof attaching and releasing therapeutic agents described herein.Liposomes are microscopic spherical lipid bilayers surrounding anaqueous core that are made from amphiphilic molecules such asphospholipids. For example, a liposome may trap a therapeutic agentbetween the hydrophobic tails of the phospholipid micelle. Water solubleagents can be entrapped in the core and lipid-soluble agents can bedissolved in the shell-like bilayer. Liposomes have a specialcharacteristic in that they enable water soluble and water insolublechemicals to be used together in a medium without the use of surfactantsor other emulsifiers. Liposomes can form spontaneously by forcefullymixing phosopholipids in aqueous media. Water soluble compounds aredissolved in an aqueous solution capable of hydrating phospholipids.Upon formation of the liposomes, therefore, these compounds are trappedwithin the aqueous liposomal center. The liposome wall, being aphospholipid membrane, holds fat soluble materials such as oils.Liposomes provide controlled release of incorporated compounds. Inaddition, liposomes can be coated with water soluble polymers, such aspolyethylene glycol to increase the pharmacokinetic half-life. Oneembodiment of the present invention contemplates an ultra high-sheartechnology to refine liposome production, resulting in stable,unilamellar (single layer) liposomes having specifically designedstructural characteristics. These unique properties of liposomes, allowthe simultaneous storage of normally immiscible compounds and thecapability of their controlled release.

In some embodiments, the present invention contemplates cationic andanionic liposomes, as well as liposomes having neutral lipids.Preferably, cationic liposomes comprise negatively-charged materials bymixing the materials and fatty acid liposomal components and allowingthem to charge-associate. Clearly, the choice of a cationic or anionicliposome depends upon the desired pH of the final liposome mixture.Examples of cationic liposomes include lipofectin, lipofectamine, andlipofectace.

One embodiment of the present invention contemplates a medium comprisingliposomes that provide controlled release of at least one therapeuticagent. Preferably, liposomes that are capable of controlled release: i)are biodegradable and non-toxic; ii) carry both water and oil solublecompounds; iii) solubilize recalcitrant compounds; iv) prevent compoundoxidation; v) promote protein stabilization; vi) control hydration; vii)control compound release by variations in bilayer composition such as,but not limited to, fatty acid chain length, fatty acid lipidcomposition, relative amounts of saturated and unsaturated fatty acids,and physical configuration; viii) have solvent dependency; iv) havepH-dependency and v) have temperature dependency.

The compositions of liposomes are broadly categorized into twoclassifications. Conventional liposomes are generally mixtures ofstabilized natural lecithin (PC) that may comprise syntheticidentical-chain phospholipids that may or may not contain glycolipids.Special liposomes may comprise: i) bipolar fatty acids; ii) the abilityto attach antibodies for tissue-targeted therapies; iii) coated withmaterials such as, but not limited to lipoprotein and carbohydrate; iv)multiple encapsulation and v) emulsion compatibility.

Liposomes may be easily made in the laboratory by methods such as, butnot limited to, sonication and vibration. Alternatively,compound-delivery liposomes are commercially available. For example,Collaborative Laboratories, Inc. are known to manufacture customdesigned liposomes for specific delivery requirements.

Microspheres, Microparticles And Microcapsules

Microspheres and microcapsules are useful due to their ability tomaintain a generally uniform distribution, provide stable controlledcompound release and are economical to produce and dispense. Preferably,an associated delivery gel or the compound-impregnated gel is clear or,alternatively, said gel is colored for easy visualization by medicalpersonnel.

Microspheres are obtainable commercially (Prolease®, Alkerme's:Cambridge, Mass.). For example, a freeze dried medium comprising atleast one therapeutic agent is homogenized in a suitable solvent andsprayed to manufacture microspheres in the range of 20 to 90 sm.Techniques are then followed that maintain sustained release integrityduring phases of purification, encapsulation and storage. Scott et al.,Improving Protein Therapeutics With Sustained Release Formulations,Nature Biotechnology, Volume 16:153-157 (1998).

Modification of the microsphere composition by the use of biodegradablepolymers can provide an ability to control the rate of therapeutic agentrelease. Miller et al., Degradation Rates of Oral Resorbable Implants(Polylactates and Polyglycolates: Rate Modification and Changes inPLA/PGA Copolymer Ratios, J. Biomed. Mater. Res., Vol. II:711-719(1977).

Alternatively, a sustained or controlled release microsphere preparationis prepared using an in-water drying method, where an organic solventsolution of a biodegradable polymer metal salt is first prepared.Subsequently, a dissolved or dispersed medium of a therapeutic agent isadded to the biodegradable polymer metal salt solution. The weight ratioof a therapeutic agent to the biodegradable polymer metal salt may forexample be about 1:100000 to about 1:1, preferably about 1:20000 toabout 1:500 and more preferably about 1:10000 to about 1:500. Next, theorganic solvent solution containing the biodegradable polymer metal saltand therapeutic agent is poured into an aqueous phase to prepare anoil/water emulsion. The solvent in the oil phase is then evaporated offto provide microspheres. Finally, these microspheres are then recovered,washed and lyophilized. Thereafter, the microspheres may be heated underreduced pressure to remove the residual water and organic solvent.

Other methods useful in producing microspheres that are compatible witha biodegradable polymer metal salt and therapeutic agent mixture are: i)phase separation during a gradual addition of a coacervating agent; ii)an in-water drying method or phase separation method, where anantiflocculant is added to prevent particle agglomeration and iii) by aspray-drying method.

In one embodiment, the present invention contemplates a mediumcomprising a microsphere or microcapsule capable of delivering acontrolled release of a therapeutic agent for a duration ofapproximately between 1 day and 6 months. In one embodiment, themicrosphere or microparticle may be colored to allow the medicalpractitioner the ability to see the medium clearly as it is dispensed.In another embodiment, the microsphere or microcapsule may be clear. Inanother embodiment, the microsphere or microparticle is impregnated witha radio-opaque fluoroscopic dye.

Controlled release microcapsules may be produced by using knownencapsulation techniques such as centrifugal extrusion, pan coating andair suspension. Such microspheres and/or microcapsules can be engineeredto achieve desired release rates. For example, Oliosphere® (Macromed) isa controlled release microsphere system. These particular microsphere'sare available in uniform sizes ranging between 5-500 μm and composed ofbiocompatible and biodegradable polymers. Specific polymer compositionsof a microsphere can control the therapeutic agent release rate suchthat custom-designed microspheres are possible, including effectivemanagement of the burst effect. ProMaxx® (Epic Therapeutics, Inc.) is aprotein-matrix delivery system. The system is aqueous in nature and isadaptable to standard pharmaceutical delivery models. In particular,ProMaxx® are bioerodible protein microspheres that deliver both smalland macromolecular drugs, and may be customized regarding bothmicrosphere size and desired release characteristics.

In one embodiment, a microsphere or microparticle comprises a pHsensitive encapsulation material that is stable at a pH less than the pHof the internal mesentery. The typical range in the internal mesenteryis pH 7.6 to pH 7.2. Consequently, the microcapsules should bemaintained at a pH of less than 7. However, if pH variability isexpected, the pH sensitive material can be selected based on thedifferent pH criteria needed for the dissolution of the microcapsules.The encapsulated compound, therefore, will be selected for the pHenvironment in which dissolution is desired and stored in a pHpreselected to maintain stability.

Examples of pH sensitive material useful as encapsulants are Eudragit®L-100 or S-100 (Rohm GMBH), hydroxypropyl methylcellulose phthalate,hydroxypropyl methylcellulose acetate succinate, polyvinyl acetatephthalate, cellulose acetate phthalate, and cellulose acetatetrimellitate. In one embodiment, lipids comprise the inner coating ofthe microcapsules. In these compositions, these lipids may be, but arenot limited to, partial esters of fatty acids and hexitiol anhydrides,and edible fats such as triglycerides. Lew C. W., Controlled-Release pHSensitive Capsule And Adhesive System And Method. U.S. Pat. No.5,364,634 (herein incorporated by reference).

In one embodiment, the present invention contemplates a microparticlecomprising a gelatin, or other polymeric cation having a similar chargedensity to gelatin (i.e., poly-L-lysine) and is used as a complex toform a primary microparticle. A primary microparticle is produced as amixture of the following composition: i) Gelatin (60 bloom, type A fromporcine skin), ii) chondroitin 4-sulfate (0.005%-0.1%), iii)glutaraldehyde (25%, grade 1), and iv)1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDChydrochloride), and ultra-pure sucrose (Sigma Chemical Co., St. Louis,Mo.). The source of gelatin is not thought to be critical; it can befrom bovine, porcine, human, or other animal source. Typically, thepolymeric cation is between 19,000-30,000 daltons. Chondroitin sulfateis then added to the complex with sodium sulfate, or ethanol as acoacervation agent.

Following the formation of a microparticle, a therapeutic agent isdirectly bound to the surface of the microparticle or is indirectlyattached using a “bridge” or “spacer”. The amino groups of the gelatinlysine groups are easily derivatized to provide sites for directcoupling of a compound. Alternatively, spacers (i.e., linking moleculesand derivatizing moieties on targeting ligands) such as avidin-biotinare also useful to indirectly couple targeting ligands to themicroparticles. Stability of the microparticle is controlled by theamount of glutaraldehyde-spacer crosslinking induced by the EDChydrochloride. A controlled release medium is also empiricallydetermined by the final density of glutaraldehyde-spacer crosslinks.

In one embodiment, the present invention contemplates microparticlesformed by spray-drying a composition comprising fibrinogen or thrombinwith a therapeutic agent. Preferably, these microparticles are solubleand the selected protein (i.e., fibrinogen or thrombin) creates thewalls of the microparticles. Consequently, the therapeutic agents areincorporated within, and between, the protein walls of themicroparticle. Heath et al., Microparticles And Their Use In WoundTherapy. U.S. Pat. No. 6,113,948 (herein incorporated by reference).Following the application of the microparticles to living tissue, thesubsequent reaction between the fibrinogen and thrombin creates a tissuesealant thereby releasing the incorporated compound into the immediatesurrounding area.

One having skill in the art will understand that the shape of themicrospheres need not be exactly spherical; only as very small particlescapable of being sprayed or spread into or onto a surgical site (i.e.,either open or closed). In one embodiment, microparticles are comprisedof a biocompatible and/or biodegradable material selected from the groupconsisting of polylactide, polyglycolide and copolymers oflactide/glycolide (PLGA), hyaluronic acid, modified polysaccharides andany other well known material.

EXPERIMENTAL

Animal protocols for cell cultures and brain slices were approved byPennsylvania State University IACUC in accordance with the NationalInstitutes of Health Guide for the Care and use of Laboratory Animals.For in vivo experiments on adult mice or neonatal rats, all procedureswere approved by the Committee of Animal Use for Research and Educationof Fudan University or South China Normal University, respectively, inaccordance with the ethical guidelines for animal research. Animal roomswere automatically controlled at 12 hr light/dark cycle, and water andfood were available ad libitum.

Example I Cell Culture And Transfection

Mouse cortical neurons were prepared from newborn C57BL/6 mice aspreviously described. Qi et al., “Cyclothiazide induces robustepileptiform activity in rat hippocampal neurons both in vitro and invivo” The Journal Of Physiology 571:605-618 (2006). Briefly, the newbornmouse cerebral cortices were dissected out in ice-cold HEPES-bufferedsaline solution, washed and digested with 0.05% trypsin-EDTA at 37° C.for 20 min. After deactivation of trypsin with serum-containing medium,cells were centrifuged, resuspended, and seeded on a monolayer ofcortical astrocytes at a density of 10,000 cells/cm² in 24-well plates.The neuronal culture medium contained MEM (500 ml, Invitrogen), 5% fetalbovine serum (Atlanta Biologicals), 10 ml B-27 supplement (Invitrogen),100 mg NaHCO₃, 2 mM Glutamax (Invitrogen), and 25 units/ml penicillin &streptomycin. AraC (4 M, Sigma) was added to inhibit the excessiveproliferation of astrocytes. Cell cultures were maintained in a 5%CO₂-humidified incubator at 37° C. for 14-21 days.

Human embryonic kidney (HEK) 293T cells were maintained in DMEMsupplemented with 10% FBS and 25 units/ml penicillin/streptomycin. PEIkit (molecular weight 25,000, Polysciences, Inc.) was applied for HEKcell transfection. In brief, 1 μg DNA was diluted into 50 μl of OptiMEM(Invitrogen), then mixed with 4 μl of PEI (1 μg/p), incubated for 5 min,and added drop-by-drop to the culture well containing 500 s of medium.After 5 hr incubation, the transfection reagents were washed off byfresh culture medium. Two days after transfection, HEK293T cells wereused for electrophysiological study. Rat CLC-3 short transcript fused toeGFP plasmid (pCLC3sGFP) was purchased from Addgene (plasmid #52423). Liet al., “The ClC-3 chloride channel promotes acidification of lysosomesin CHO-K1 and Huh-7 cells” American Journal Of Physiology” CellPhysiology 282:C1483-1491 (2002).

Example II Cell Viability Assay

A LIVE/DEAD® Viability/Cytotoxicity Assay Kit (L3224, Life Technologies)containing ethidium homodimer-1 and calcein-AM was used to examine cellviability. Ethidium homodimer-1 binds to cellular DNA and typicallylabels dead cells in red fluorescence, while Calcein-AM can be cleavedby esterases in live cells to give strong green fluorescence. After drugtreatment, neurons were incubated in bath solution containing 1 μMcalcein-AM and 4 μM ethidium homodimer-1 at room temperature for 40 min.Cell survival and death rate were measured by quantifying the percentageof green and red fluorescent cells, respectively. For each group, atleast 5 fields of each coverslip were imaged for data analysis.

Example III Mouse Brain Slice Preparation

Brain slices were prepared from C57BL/6 mice (male and female). Animalswere anesthetized with Avertin (tribromoethanol, 250 mg/kg) anddecapitated. Hippocampal horizontal sections (400 μm) were prepared byLeica VT1200S vibratome in ice-cold artificial cerebral spinal fluid(aCSF) (in mM): 125 NaCl, 26 NaHCO₃, 10 glucose, 2.5 KCl, 2.5 CaCl₂,1.25 NaH₂PO₄, and 1.3 MgSO₄, osmolarity 290-300 mOsm, aerated with 95%O₂/5% CO₂. Slices were then transferred to incubation chamber containingnormal aCSF saturated with carbogen (95% O₂/5% CO₂) at 33° C. for 30min, followed by recovery at room temperature for 1 hour before use.Individual slice was transferred to a submerged recording chamber wherethey were continuously perfused (2-3 ml/min) with aCSF saturated by 95%O₂/5% CO₂ at 31-33° C. (TC-324B, Wamer instruments Inc). Slices werevisualized with infrared optics using an Olympus microscope equippedwith DIC optic.

Example IV Electrophysiology Cell Culture

The cultured neurons were placed in the recording chamber withcontinuous perfusion of the bath solution consisting of (mM): 128 NaCl,10 Glucose, 25 HEPES, 5 KCl, 2 CaCl₂, 1 MgSO₄, pH 7.3 adjusted withNaOH, and osmolarity 300 mOsm. For recording spontaneous firing undercurrent clamp mode, pipettes were filled with an internal solutioncontaining (in mM): 125 K-gluconate, 5 Na-phosphocreatine, 5 EGTA, 10KCl, 10 HEPES, 4 Mg-ATP, 0.3 Na-GTP, 280-290 mOsm, pH 7.3 adjusted withKOH. Epileptiform activity in cultured neurons was induced either by 10μM CTZ for 24 h, or 1 μM KA for 2 h, or 50 μM 4-AP for 2 h. The burstactivity was defined as previously described. Qi et al., “Cyclothiazideinduces robust epileptiform activity in rat hippocampal neurons both invitro and in vivo” The Journal Of Physiology 571:605-618 (2006). Inbrief, at least five consecutive action potentials overlaying on top ofthe large depolarization shift (210 mV depolarization and >300 ms induration).

For Cl⁻ current recording, a 0 Ca2+ pipette solution contained (mM): 125CsCl, 5 Na-phosphocreatine, 10 HEPES, 5 EGTA, 5 TEACl, 4 MgATP (pH 7.3adjusted with CsOH, 280-290 mOsm).

To isolate Cl⁻ current: (1) Extracellular Na was replaced by NMDG⁺ andvoltage-dependent Na⁺ channels were blocked by tetrodotoxin (TTX); (2)K⁺ and Ca²⁺ were removed from bath solution; (3) K⁺ channels wereblocked with Cs⁺ and tetraethylamonium (TEA) in the pipette solution,and 4-AP in the bath solution; (4) CdCl₂ was added into bath solution toblock Ca²⁺ channels; (5) Picrotoxin (50 μM) was also included to blockGABAA receptors. Thus, the external solution contained the following (inmM): 135 NMDG-Cl, 20 HEPES, 20 Glucose, 5 4-AP and 2 MgSO₄, supplementedwith 1 μM TTX, 200 μM CdCl₂, and 50 μM picrotoxin, osmolarity 300 mOsm,pH7.3-7.4 after aerated with 95% O₂/5% CO₂. Voltage steps from −80 to+90 mV (10 mV increments) were applied from holding potential of 0 mV(ECl⁻˜0 mV). Data were collected with a MultiClamp 700A amplifier andpCLAMP9 software (Molecular Devices).

Brain Slice Recording

Field potential recordings were performed with glass electrodes (2-4 MStip resistance) filled with external solution. The glass electrode wasplaced into the CA3 pyramidal layer. For current clamp (I=0) recordings,the amplifier was set at the 100× with a band pass filter of 0.1-5 KHz.All recordings were performed at 31-33° C. The epileptic activity wasevoked by Mg²⁺-free aCSF, or addition of 50 μM 4-AP or 8.5 mM K+ in theaCSF.

A Hamming window function was applied before power spectrum analysis.Power was calculated by integrating the root mean square value of thesignal in frequency bands from 0.1 to 1000 Hz in sequential 5-min timewindows before, during, and after drug applications. To avoidslice-to-slice and electrode contact variability, power values werenormalized to control condition before drug application for each slice,and then averaged across different slices for statistical analysis.

Example V Immunostaining And Western-Blot

Brain slices were prepared following the electrophysiology protocoldescribed above, with 200 μm thickness for immunostaining and 400 μmthickness for Western-blot. In general, slices were recovered for 1 hrat room temperature and then randomly divided into two groups. One groupof slices was incubated in normal aCSF for 1 hr at 33° C. Another groupof slices were incubated in 0 Mg²⁺ aCSF for 1 hr at 33° C. Slices werethen fixed by 4% PFA overnight at 4° C.

Immunostaining

For CLC-3 staining, slices were pretreated with blocking solution (0.3%Triton-X and 5% normal donkey and goat serum in 0.1 M PBS) for 2 hr, andthen incubated for 72 hr with CLC-3 primary antibody (Rabbit, 1:200,Alomone, ACL-001). Some slices were incubated with rabbit IgG ascontrol. After washing three times in PBS with 0.01% triton-X, the brainsections were incubated with goat anti rabbit secondary antibodiesconjugated to Cy3 (1:500, Jackson ImmunoResearch) for 2 hr at roomtemperature. The brain sections were mounted on a glass slide with ananti-fading mounting solution (Invitrogen). Fluorescent images wereacquired on an Olympus confocal microscope system (FV1000). For eachslice, at least 2-3 fields of CA3 were imaged. To quantify CLC3fluorescent intensity in CA3 pyramidal layer, confocal images wereanalyzed using NIH Image J software.

Western Blot

400 μm thickness hippocampal slices were randomly divided into twogroups after recovery for 1 hr at room temperature, which were the sameas described above for immunostaining. The hippocampus was dissected outin ice-cold aCSF. Proteins were extracted with RIPA lysis buffer(containing 1:100 Phenylmethylsulfonyl fluoride and 1:200 Proteaseinhibitor). Then, add 1× loading dye buffer (NuPAGE LDS sample buffer(4×)) and 1% beta-ME into the protein sample, and mix thoroughly.Protein extracts (2 μg/p, 15 μl per lane) were boiled at 55° C. for 20min, loaded on 10% gel, separated using sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) (70V for 50 min thenswitch to 90V for 2 hrs) at room temperature. Then the gel was blottedonto a PVDF membrane. Blots were blocked using a solution of 5% milk in1% Tween-20 in Tris-buffered solution (1×TBS) for 1 hr at roomtemperature. The primary antibodies to CLC3 (Rabbit, 1:300, Alomone,ACL-001) and GAPDH (Rabbit, 1:20000, Sigma, G9545) were appliedovernight on a rotator at 4° C. in blocking solution containing 5% milkand 1% Tween-20 in 1×TBS. Blots were then incubated with anti-rabbitsecondary antibodies at 1:20000 dilution for 1.5 hrs at roomtemperature, washing three times using 1×TBS, and then imaging thesignal with the Odyssey scanner system (Li-Cor Biosciences).

Example VI Electroencephalogram (EEG) Recording

To test the in vivo anti-epilepsy effects of gluconate in neonatal andadult rodents, P8-12 Sprague-Dawley rats or 2-month old male C57BL/6mice were deeply anesthetized with pentobarbital sodium (50 mg/kg forneonatal rat and 100 mg/kg for adult mice, intraperitoneal injection).Two stainless steel screws (1 mm in diameter) were inserted in the skullabove the cortex as EEG recording electrodes, one ground electrode aswell as one reference electrode were located +1.8 mm anterior to bregma,+0.5 mm lateral to the midline, and 1 mm below the cortical surface. Allelectrodes were attached to a micro-connector and fixed onto the skullwith dental cement. After surgery, neonatal rats were returned to theirmothers and allowed to recover for 2 days prior to subsequent EEGrecording. The adult mice were single housed in order to prevent damageof the implanted electrodes and allowed to recover at least 5 daysbefore EEG recording.

The baseline of EEG was recorded for 0.5-1 hr to allow the animal toadapt to the environment. Kainic acid (2 mg/kg) was administeredintraperitoneally (i.p.) for neonatal epilepsy induction. D-gluconicacid sodium salt (2 g/kg) or 0.9% saline was injected i.p. 10 min afterKA administration. Pentylenetetrazol (PTZ) was administeredintraperitoneally for inducing a stable epileptic burst activity inadult mice, and D-gluconic acid sodium salt (1 g/kg and 2 g/kg) or 0.9%saline was injected i.p. 10 min before PTZ administration. Epileptiformactivity was monitored for 1 hr after PTZ injection. Qian et al.,“Epileptiform response of CA1 neurones to convulsant stimulation bycyclothiazide, kainic acid and pentylenetetrazol in anaesthetized rats”Seizure 20:312-319 (2011). After the experiment, animals were injectedwith diazepam to protect the animals from recurrent epilepsy.

The electrophysiological signals were amplified (1000×) and filtered(0-500 Hz) by using a NeuroLog System (Digitimer Ltd, Hearts, UK) andvisualized and stored in a PC through a D-A converter, CED 1401 micro(Cambridge Electronic Design, Cambridge, UK).

Analysis of EEG

The power level of different frequency components in the neonatal EEGsignal was revealed by power spectrum analysis. Power was calculated in1-min time windows by integrating the power of dominant frequency bandsfrom 1 to 100 Hz (EEG band).

Adult EEG signal were analyzed by spike 2 software (an analysis programfor CED 1401, Cambridge, UK). The EEG spike was defined as exceedingtwice of the baseline amplitude, and the EEG burst was defined as havinghigh frequency (>1 Hz) and high amplitude multiple spikes lasting morethan 10 s. The latency of spike or burst was defined as the time fromPTZ injection to either the first spike or the first burst.

Example VII Seizure Behavior Test

Male mice were divided into three groups: vehicle control group (saline,n=9), D-gluconic acid sodium at 1 g/kg group (n=10), and D-gluconic acidsodium at 2 g/kg group (n=11). PTZ (50 mg/kg) was intraperitoneallyadministered 1 hr after drug treatment. After PTZ injection, thebehavior was monitored and observed for 1 hr. The seizure behavior wasclassified according to Racine Score: stage 0, no response; stage 1,facial twitching; stage 2, nod; stage 3, forelimb clonic, tail upright;stage 4, stand with forelimbs clonic; stage 5, stand and fall, jump,general clonic seizure, general clonic and tonic seizure or death.Racine et al., “Modification of seizure activity by electricalstimulation. 3. Mechanisms” Electroencephalogr Clin Neurophysiol32:295-299 (1972).

Example VIII Data Analysis

Data were shown as mean±s.e.m. Student's t-test (paired or unpaired) wasperformed for two-group comparison, and the χ2 test was used to comparethe difference of percentage between two groups. For comparison amongmultiple groups, one-way ANOVA with post hoc tests were used.Statistical significance was set at P<0.05.

Example IX Epilieptiform Activity Reduction By Glucose Oxidase

This example shows preliminary data demonstrating that glucose oxidasecan effectively inhibit epileptiform activity.

Hippocampal slices were prepared from neonatal mice. Epileptiformactivity was elicited by Mg²⁺-free aCSF (artificial cerebral-spinalfluid) or high K⁺ (8.5 mM) aCSF. Glucose oxidase (GOx) was eitheracutely applied to the slices or pre-incubated for 90 min in aCSF beforeapplication to the slices.

The data shows that glucose oxidase dose-dependently inhibited theepileptiform activity induced by 0 Mg2+ aCSF. FIG. 18A. Glucose oxidasewas also seen to inhibit the epileptiform activity induced by high K+.FIG. 18B.

Different glucose concentrations ranging from 4 mM to 20 mM were alsotested. When 1 unit glucose oxidase/ml was provided an inhibition ofepileptiform activity was observed. FIG. 19A. In constrast, an acuteapplication of 0.1 unit glucose oxidase/ml did not have significanteffect. FIG. 18A, top row. However, after a ninety (90) minutepre-incubation 0.1 U/ml glucose oxidase also inhibited epileptiformactivity. FIG. 19B.

These results suggested that glucose oxidase may be useful as anantiepileptic drug.

1.-4. (canceled)
 5. An oral pharmaceutical composition comprisingglucose oxidase and supplementary glucose, wherein said composition actsto reduce convulsion activity in a neonate having an upregulated CLC-3Cl⁻ channel compared to a non-neonate.
 6. The oral pharmaceuticalcomposition of claim 5, wherein said neonate has circulating glucose. 7.The oral pharmaceutical composition of claim 6, wherein said glucoseoxidase coverts said circulating glucose to gluconate.
 8. The oralpharmaceutical composition of claim 7, wherein said gluconate inhibitssaid CLC-3 Cl⁻ channel.
 9. The oral pharmaceutical composition of claim8, wherein inhibition of said CLC-3 Cl⁻ channel reduced said convulsionactivity in said neonate.
 10. The oral pharmaceutical composition ofclaim 5, wherein said neonate is diagnosed with neonatal epilepsy. 11.The oral pharmaceutical composition of claim 5, wherein said neonate isless than 1 month old.
 12. The oral pharmaceutical composition of claim5, wherein said composition further comprises a pharmaceuticallyacceptable carrier.
 13. A pharmaceutical formulation for the use intreating convulsion activity in a neonate, wherein said formulationcomprises a fixed dose of a CLC-3 Cl⁻ channel inhibitor, supplementalglucose, and an inactive carrier.
 14. The pharmaceutical formulation ofclaim 13, wherein said CLC-3 Cl⁻ channel inhibitor is glucose oxidase.15. The pharmaceutical formulation of claim 13, wherein said inactivecarrier is saline.
 16. The pharmaceutical formulation of claim 14,wherein said neonate has circulating glucose and said glucose oxidaseconverts said circulating glucose to gluconate.
 17. The pharmaceuticalformulation of claim 14, wherein said supplemental glucose and saidglucose oxidase is administered at said fixed dose of a glucose infusionrate of 7.3 mg/kg/minute.
 18. The pharmaceutical formulation of claim17, wherein said administered is by intravenous injection.
 19. Thepharmaceutical formulation of claim 13, wherein said neonate isdiagnosed with neonatal epilepsy.
 20. The pharmaceutical formulation ofclaim 13, wherein said neonate has an upregulated CLC-3 Cl⁻ channelcompared to a non-neonate.
 21. The pharmaceutical formulation of claim13, wherein said neonate is less than 1 month old.
 22. Thepharmaceutical formulation of claim 13, wherein said pharmaceuticalformulation is administered by a transdermal patch.